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本研究建立了一种检测传染性造血器官坏死病毒(IHNV)的新型荧光环介导等温扩增法,为实验室及现场检测IHNV提供特异性强、灵敏度高、快速、能实时观察扩增荧光信号的检测技术。针对IHNV全基因组序列设计了一组6条LAMP特异性引物,对4株不同来源的IHNV和8株不同的病毒进行特异性实验和灵敏度实验,结果表明该方法具有高度特异性,检测灵敏度可达3.64×10-7μg/μL,比常规RT-PCR高1000倍,与荧光RT-PCR方法相当。通过环介导等温扩增技术(LAMP)和荧光检测技术相结合,使其检测时间能缩短至15~20分钟内完成检测,并可杜绝由于开盖加染料而导致的假阳性率偏高问题,在鱼病快速检测上具有重要意义。
In this study, a novel fluorescence ring-mediated isothermal amplification method for the detection of infectious hematopoietic necrosis virus (IHNV) was established to provide IHNV with high specificity, sensitivity, and fastness for laboratory and on-site detection of fluorescence amplification in real time Signal detection technology. A set of six LAMP-specific primers designed for IHNV genome-wide sequence was used to test the specificity and sensitivity of four different strains of IHNV and eight different viruses. The results showed that the method is highly specific and has good detection sensitivity 3.64 × 10-7μg / μL, which is 1000 times higher than that of conventional RT-PCR, which is equivalent to fluorescence RT-PCR method. By ring-mediated isothermal amplification (LAMP) and fluorescence detection technology, the detection time can be shortened to 15 to 20 minutes to complete the test, and can eliminate the false positive rate due to open the lid plus dye problem It is of great importance in the rapid detection of fish diseases.