【摘要】 为提高大黄鱼刺激隐核虫病的病原体检出率,本研究针对刺激隐核虫(Cryptocaryon irritans)18S-ITS2基因序列设计4条LAMP引物,在内外引物浓度比2∶1～6∶1、Mg2+浓度2 mmol/L、反应温度61℃～66℃、反应时间60 min的优化条件下,扩增产物经电泳后呈现特异性的LAMP梯形条带。本研究建立的LAMP方法具有高灵敏度(10-7ng/μL DNA浓度),比常规PCR方法高100倍。将该方法应用于采集自不同地域的虫体样本、病鱼和水样以及不同组织样品的检测,结果在13份样品中有10份检出阳性,并显示该方法既能够检测发病鱼,也能够检出已感染但未发病的鱼。实验表明,该方法是一种能够快速、简易、特异、敏感地检测海水鱼类刺激隐核虫病的病原学检测方法。更多还原

【Abstract】 To improve the detection rate of Cryptocaryon irritans on Pseudosciaena crocea(Richardson).A set of four primers were designed based on the sequence of the 18S-ITS2 of C.irritans for the use of the loop-mediated isothermal amplification(LAMP) assay.The assay conditions was optimized as following: 2∶1 to 6∶1 inner primer concentration ratio,2 mmol/L of Mg2+,at 61 ℃ to 66 ℃ for 60 min.LAMP amplification products had ladder-like bands when electrophoresed on an agarose gel.The detection limit of the assay was about 10-7 ng/μL of C.irritans DNA.Hence,the sensitivity of LAMP assay was 100-fold higher than the standard PCR protocol.The assay was applied to detact the presence of C.irritans in parasite,diseased fish and water samples collected from different region,as well as different tissue samples.Ten positive were detected in 13 samples.This study showed LAMP assay could be used not only to diagnose the infected fishes,but also to detect the carrier of C.irritans as well.The LAMP assay is a rapid,simple,specific and sensitive test to diagnose for marine fish pathogenic C.irritans.更多还原