碩士 / 國立高雄海洋科技大學 / 海洋生物技術研究所 / 104 / The outbreak of acute hepatopancreatic necrosis disease (AHPND) from 2009 has caused mass losses in shrimp farms worldwide. The strain of Vibrio parahaemolyticus which obtain a plasmid and secret toxins has been determined as the infectious agent. PCR methods were published and used to perform diagnosis in laboratory. This study developed methods of Loop-mediated isothermal amplification (LAMP) and Recombinase polymerase amplification (RPA) to detect AHPND and aimed to carry out the diagnosis at the farm side. Compared to PCR, the nucleic acid amplification of LAMP and RPA is performed at a constant temperature which shortens the reaction time and excludes complex equipments. The results are directly color presented by fluorescent dye or lateral flow dipstick which substitute for the gel electrophoresis. The LAMP system for detection of AHPND have six primers targeting toxin A gene (333 bp) in the plasmid under 65 ℃condition. LAMP results were observed through the stain of fluorescent dye and showed the color change emitted by ultraviolet light. RPA have designed two methods for amplification of the target sequence, one is using two labeled primers, the other is added the probe to increase the specificity. After reaction at 37 ℃, both methods followed with lateral flow dipstick which antibody labeled recognize RPA amplicons. Combined with the DNA easy extraction method for template preparation, the time needed to complete the diagnosis is only 1-2 hours. The sensitivity and specificity of LAMP and RPA are better or comparable to PCR in this study. We also examined several white shrimps (Litopenaeus vannamei) from Kaohsiung and found positive signals. It revealed the distribution and consideration of AHPND.
| Identifer | oai:union.ndltd.org:TW/104NKIM0270005 |
| Date | January 2016 |
| Creators | DAI,NING, 戴寧 |
| Contributors | Tsai,Jyh-Ming, 蔡志明 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 51 |
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