同时检测贝类派琴虫和包纳米虫的双重LAMP方法的建立

doi:10.16431/j.cnki.1671-7236.2015.08.003

Abstract

Abstract: In order to establish a duplex loop-mediated isothermal amplification (LAMP) method for the detection of shellfish parasitic infection with Perkinsus and Bonamia, primers were designed according to the conserved region of ITS and 18S rRNA gene sequences of Perkinsus and Bonamia deposited in GenBank using the online Primer Explorer 4.0.Firstly, the respective single LAMP detection assay of two parasites were developed, and then the reaction conditions were optimized by combining the two reaction systems into duplex LAMP method.The specificity and sensitivity of duplex LAMP method were examined and the confidence was evaluated by digesting the amplicons with respective endonuclease.The results showed that the established duplex LAMP method was specific enough to distinguish the two parasites from other parasites, and the detection limit of Perkinsus was 10 copies/μL plasmid DNA, the detection limit of Bonamia was 100 copies/μL plasmid DNA.The endonuclease digestion of duplex LAMP products certified the confidence of the established duplex LAMP method.The detection result of 20 Ruditapes philippinarum samples collected randomly from the East China sea and Yellow sea and 10 Ostrea edulis samples imported from USA showed that the duplex LAMP method could be used as a rapid detection method for infection with Perkinsus and Bonamia.

Key words: shellfish parasitic disease; loop-mediated isothermal amplification (LAMP); detection; Perkinsus; Bonamia

CLC Number:

WANG Cai-xia, FENG Chun-yan, WANG Qiao-li, YUAN Xiang-fen, DENG Jun-hua, LV Ji-zhou, WU Shao-qiang. Establishment of Duplex Loop-mediated Isothermal Amplification Method for Simultaneous Detection of Shellfish Infected with Perkinsus and Bonamia[J]. , 2015, 42(8): 1935-1942.

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Establishment of Duplex Loop-mediated Isothermal Amplification Method for Simultaneous Detection of Shellfish Infected with Perkinsus and Bonamia

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