环介导恒温扩增技术快速检测短凯伦藻

短凯伦藻(Karenia brevis)是一种有毒赤潮微藻,所产生的短藻毒素对海洋生物乃至人类都有毒害作用,为加强对短凯伦藻赤潮的监控,建立了稳定的短凯伦藻环介导恒温扩增(LAMP)鉴定体系,在此基础上进行了特异性和灵敏性验证.特异性实验结果显示只有短凯伦藻或者含短凯伦藻的模板呈现阳性反应,其他微藻为阴性反应,从而验证了该LAMP方法的特异性;同时,对短凯伦藻的基因组DNA进行一系列10倍稀释作为敏感度实验的模板,并与常规PCR做了对比,结果表明:短凯伦藻的LAMP方法最低检测限度为50 pg,敏感度比常规PCR高10倍.LAMP产物鉴定不需要常规的胶电泳过程,直接采用肉眼观察的方法,在含有短凯伦藻的阳性反应管中会出现白色混浊,加入SYBRòGreen I染料呈现绿色,而未含有短凯伦藻的阴性管为澄清,染色后仍为原来的橙色.因此,该方法操作简便、特异性强、灵敏度高而成本低,在赤潮原因种检测监控方面具有良好的应用前景.

Rapid Detection of Karenia brevis (Dinophyceae) by Loop-mediated Isothermal Amplification (LAMP)

Karenia brevis(Gymnodinium breve) is an unarmored,non-peridinin-containing dinoflagellate.It produces lipophilic brevetoxin that can harm many marine animals,eg.fish,birds and shellfish.As for human,brevetoxin can cause respiratory distress by inhalation and food poisoning by consumption of contaminated shellfish.Previous methods for the detection of K.brevis depend on microscopy analysis,which is time-consuming and requires a considerable amount of expertise and skill.Molecular methods have also been applied to detect K.brevis,e.g.real-time PCR and DNA hybridization assay.However,there are some inevitable disadvantages for the two methods,such as expensive reagents and equipment,or fussy approaches.Loop-mediated isothermal amplification(LAMP) is a specific nucleic acid amplification method that is easy to perform.The LAMP method can amplify nucleic acids under isothermal conditions at approximately 65 ℃.As a result,it allows for the use of simple and inexpensive reaction equipment.The identification system of microalgae K.brevis was successfully established in this study using LAMP method.The specificity and sensitivity were validated based on this detection system.Nine different algae were investigated to confirm the specificity of the LAMP for detecting K.brevis.The results showed that only K.brevis and the samples containing K.brevis were amplified;no amplification occurred in all the other samples including K.mikimotoi.Moreover,LAMP was able to detect template at a 100 to 10-3 dilution,whereas regular PCR could amplify the sample at only a 100 to 10-2 dilution.Therefore,the detection limit of the LAMP system was approximately 5.0×10-2 ng(50 pg) DNA,which was 10 times more sensitive than conventional PCR.LAMP products could be identified by adding SYBR Green I dyes or by the production of a white precipitate.This visual inspection has predominance because there is no need for gel electrophoresis and staining with ethidium bromide.So this method illustrates a novel application prospect for forecasting red-tides caused by K.brevis because of its high sensitivity,rapid reaction and the need for only simple equipment.