全预混冻干PCR试剂检测草鱼呼肠孤病毒Ⅱ型的研究

草鱼呼肠孤病毒Ⅱ型是导致草鱼出血病的主要病原体之一,目前在我国广泛流行。本研究根据GenBank中草鱼呼肠孤病毒Ⅱ型基因序列,设计了草鱼呼肠孤病毒Ⅱ型特异性探针和PCR引物,通过优化反应条件,建立了草鱼呼肠孤病毒Ⅱ型一步法TaqMan荧光定量RT-PCR检测方法。试验对反转录和PCR反应体系进行摸索,通过优化的冻干工艺制备出草鱼呼肠孤病毒Ⅱ型全预混RT-PCR试剂,在荧光定量PCR中,冻干试剂的检测灵敏度可达10个模板,是普通PCR的100倍。批次间变异系数小于4%。特异性试验结果表明,该方法可特异性地检出草鱼呼肠孤病毒Ⅱ型,而对鲤春病毒血症病毒、传染性造血器官坏死症病毒无检测信号。该冻干试剂4℃保存12个月,室温25℃保存3个月,37℃保存15d,敏感性仍为10个,与第1d相同;利用手持单通道荧光PCR仪,可在42min内完成核酸诊断。本试验基于荧光定量PCR检测方法,结合核酸扩增体系冻干工艺制备的全预混草鱼呼肠孤病毒Ⅱ型RT-PCR试剂具有耐保存便于运输、准确性高、仪器拓展性好的特点,对草鱼呼肠孤病毒Ⅱ型快速诊断有重要意义。

Detection of Grass carp reovirus Ⅱ by Premixed Freeze-dried PCR

Grass carp reovirusⅡ(GCRVⅡ)as one of the major causative pathogens involved in grass cap hemorrhagic disease is currently widespread in China,with an urgent need for development of diagnostic products with sensitivity and convenience.In this study,GCRVⅡ-specific probes and PCR primers were designed according to the GCRVⅡgene sequences in GenBank.A TaqMan-based real-time quantitative RT-PCR assay was established by optimizing the reaction conditions.Premixed RT-PCR kits were prepared for optimizing the conditions of RT-PCR reactions as well as the freeze-dried process.This technique is shown to be highly sensitive with detection limit of nucleic acids(10 copies),100 times as normal PCR.The intra-assay trials demonstrated that the variations were less than 4%for the lyophilized reagent,indicating that it is good reproducibility.The assay was highly specific for GCRVⅡ,without cross-signals to Spring viremia of Carp virus(SVCV)and Infectious hematopoietic necrosis virus(IHNV),and the lyophilized reagent was still effective when it was stored for 12 months at 4℃,for 3 months at 25℃and for 15 days at 37℃.However,the sensitivity was still 10 copies,the same as that at the first day.The nucleic acid diagnosis can be carried using handheld single-channel fluorescence PCR instrument within 42 min.The premixed GCRVⅡRT-PCR method,combined fluorescence quantitative PCR detection,is characterized by resistance to storage and easy to transport,and will be of great significance in rapid clinical diagnosis on GCRVⅡ.