实时荧光定量PCR法检测对虾皮下和造血器官坏死病毒

应用实时荧光定量PCR最常用的TaqMan探针技术设计了探针和引物,并且用质粒技术构建了含有传染性皮下及造血器官坏死病毒(IHHNV)基因片段的阳性质控品,建立了检测IHHNVV的实时荧光定量PCR方法。对影响PCR的主要因素进行了优化,Mg2+浓度、引物和探针浓度、退火温度等均对扩增效率有明显影响。当Mg2+浓度为3.0-4.5mmol,退火温度为59～60℃时可获得最佳扩增效果。灵敏性试验表明该反应可检测体系中10拷贝的病毒核酸;该体系检测IHHNV具有很高特异性;对临床样品检测结果表明,该方法能快速、准确地检测样品中的IHHNV。

Real-Time Fluorescence Quantitative PCR for the Detection of Infectious Hypodermal and Haematopoietic Nerosis Viurs in Penaeid Shrimp

The TaqMan probe technology which was the most common technique in real-time fluorescence quantitative PCR was applied in this report. The probe and primer had been designed and the plasmid containing a partial gene of IHHNV developed from E.coli DH5α was used as positive control to examine IHHNV. The major factors affecting the reaction had been optimized and the different concentration of Mg2+ and probe and primer and the anneal temperature had obvious effects on PCR efficiency. It was proved that the optimal reaction system was 3.0 to 4.5mmol of Mg2+ and 59 to 60℃ for anneal temperature. Sensibility test showed that the method could detect 10 copies of IHHNV. It has good specifity in detecting IHHNV, and was proved as a quick and accurate method to detect clinical samples.