环介导恒温扩增技术检测血卵涡鞭虫

血卵涡鞭虫病是海水甲壳类的重要寄生虫病,其流行范围广、死亡率高、危害非常严重。本研究根据GenBank中已登录的血卵涡鞭虫(Hematodinium sp.)ITS1序列设计了1套引物,该引物可识别目标基因中6个不同区段。以此套引物建立了一种基于环介导恒温扩增技术(Loop-mediated isothermal amplification,LAMP)的血卵涡鞕虫病诊断方法。特异性试验结果表明,该套引物对血卵涡鞕虫检测具有较高的特异性,能有效检出血卵涡鞕虫。敏感度试验结果表明,该LAMP技术的灵敏度比常规PCR技术高4个数量级。分别运用LAMP和常规PCR技术对25份临床疑似病例进行检测,LAMP方法共检出感染病例25份,常规PCR检出感染病例23份。该技术能在65℃恒温条件下45～60min完成目的DNA的扩增,可直接通过肉眼观察反应产物中是否产生白色沉淀或经SYBRGreenI染色后通过颜色变化、扩增产物的琼脂糖凝胶电泳来定性判断结果,这将为血卵涡鞕虫病的临床诊断提供一种更加简便、快速、实用的方法。

Detection of Hematodinium in marine crustacean by loop-mediated isothermal amplification

is a important parasite of marine crustacean. detection system was developed by using the loop-mediated isothermal amplification (LAMP) method. A set of four primers including two outer primers and two inner primers, were designed from DNA extracted from infected crabs under constant temperature of 65 The amplification products were detected visually by ovserving whether white deposition comes into being or using SYBR green I dye, which turns green in the presence of amplified products and remains orange in its absence, or by electrophoresis. The assay was found to be 10⁴ fold more sensitive than PCR assay. A comparison between LAMP and PCR assay using 25 clinical samples showed good correlation. The developed LAMP assay is simple, rapid, cost-effective, specific and highly sensitive for disease detection.