水产学报2012，Vol.36Issue(8)：1281-1289,9.DOI:10.3724/SP.J.1231.2012.27956
奥尔森帕金虫环介导等温扩增(LAMP)检测方法的建立及应用
Establishment and application of a loop-mediated isothermal amplification (LAMP) method for Perkinsus olseni detection
摘要
Abstract
Perkinsus olseni(P. olseni) is one of the important pathogenic parasites of the shellfish. With the purpose of building a rapid, sensitive, accurate and easy to use detection method for P. olseni, we established a P. olseni loop-mediated isothermal amplification assay(LAMP) based on the internal transcribed spacer(ITS) of Perkinsus olseni 5.8S rDNA sequences. We used the online software Primer Explorer V4 (http://primerexplorer.jp/e/) and designed a set of 4 LAMP primers(Perk-FIP, Perk-BIP, Perk-F3 and Perk-B3) of the P. olseni, then we optimized the reaction conditions, mainly about the reaction temperature, magnesium ion concentration of the reaction system and the reaction time. After that, we got the P. olseni 25 uL LAMP reaction system, including: 2.5 μL l0×ThermoPol Reaction Buffer, 4 μL dNTPs(10 mmol/L each), 5 μL Betaine(5 mol/L), 1.6 uL Perk-FIP(25 umol/L), 1.6 μL Perk-BIP(25 umol/L), 1 μL Perk-B3(5 umol/L), 1 uL Perk-F3(5 umol/L), 4 uL MgCl2(25 mmol/L), 2.3 uL sterile water, 1 μL Bst DNA poly-merse(8 000 U/mL) and 1 μL DNA template, the optimal reaction temperature is 64 ℃ and the optimal reaction time is 60 min. In this research, the LAMP products were detected mainly using agarose gel electrophoresis and visual inspection of a color change due to addition of fluorescent dye. Before confirming the minimum threshold of the LAMP, we constructed P. olseni positive plasmid, also based on P. olseni 5.8S rDNA ITS sequences. The result shows that the minimum threshold of the LAMP assay is approximately 30 copies of plasmid DNA. We proved that the developed LAMP method was highly specific for P. olseni, and no cross-reaction was observed with other pathogens, such as Perkinsus marinus{P. marinus), Bonamia exitiosa(B. exitiosa), Ichthyobodo sp. and Acute Viral Necrosis Virus(AVNV). A comparative evaluation of the LAMP and PCR assays using 20 Ruditapes philippinarum(R. philippinarum) samples showed that LAMP is more sensitive and accurate than PCR and the shellfish parasite P. olseni is widely distributed in farming shellfish of North shellfish farming area. Totally, these results indicate that the LAMP method is a kind of simple, sensitive, specific, and reliable technique for the detection of P. olseni. The LAMP technique could be used for the detection of P. olseni in the coastal shellfish farms and laboratories with simple equipment.关键词
奥尔森帕金虫/内转录间隔区/环介导等温扩增/贝类/检测Key words
Perkinsus olseni/ internal transcribed spacer/ loop-mediated isothermal amplification/ shellfish/ assay分类
农业科技引用本文复制引用
曲朋,王崇明,任伟成,梁彦韬,贾志磊,黄倢,潘鲁青..奥尔森帕金虫环介导等温扩增(LAMP)检测方法的建立及应用[J].水产学报,2012,36(8):1281-1289,9.基金项目
国家“八六三”高技术研究发展计划(2006AA100307) （2006AA100307）
现代农业产业技术体系建设专项(CARS-48) （CARS-48）
