奥尔森帕金虫环介导等温扩增(LAMP)检测方法的建立及应用

摘要

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.%奥尔森帕金虫是重要的贝类病原性寄生虫之一,为建立快速、灵敏、准确和使用简便的检测方法,实验根据奥尔森帕金虫5.8S rDNA中的内转录间隔区(internal transcribedspacer,ITS)序列,建立了环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测方法,并对反应温度、反应体系中Mg2+浓度和反应时间进行了优化.该方法的检测灵敏度约为30拷贝质粒DNA,并且特异性较强,与海水帕金虫、包纳米虫、波豆虫及急性病毒性坏死病毒(acute viral necrosis virus,AVNV)等病原均无交叉反应.使用LAMP法对两批菲律宾蛤仔样品进行检测,结果表明,LAMP检测与传统PCR检测相比,灵敏度更高,检测结果更准确.实验所建立的奥尔森帕金虫LAMP检测方法简单、快速、灵敏且特异性强,可以在沿海贝类养殖厂及条件简陋的实验室使用.