摘要
根据GenBank登录的斑点叉尾鮰病毒株ORF8基因序列,设计引物和Taqman荧光探针,建立了用于检测斑点叉尾鮰（Ictalurus Punctatus）病毒的实时荧光定量PCR方法;对实时荧光定量PCR反应条件进行了优化,评估了该方法的特异性、灵敏度和重复性,并建立了绝对定量方法。特异性试验证实,该方法只能检测到斑点叉尾鮰病毒,与真鲷虹彩病毒、蛙虹彩病毒、锦鲤疱疹病毒、鳜鱼传染性脾肾坏死病毒等多种常见的水生动物病毒没有交叉反应,具有高度的特异性。标准曲线表明,在3.3×10~3.3×107拷贝数之间有较好的线性关系（r=0.998 3）,最少可检测到33个拷贝的阳性质粒,敏感度很高。同一样品于试验内及试验间的重复性试验发现,变异系数分别为0.7%和1.2%,重复性较好。该方法的检测时间从核酸抽提到出具结果仅需3h。试验结果表明,采用实时荧光PCR检测斑点叉尾鮰病毒方法具有特异性好、灵敏度高、检测耗时短的特点,并且能对病毒进行准确的定量分析,不仅适合口岸进出口检验检疫的需求,而且可以作为研究该病毒感染过程的有力工具。
To establish a specific,sensitive method of TaqMan-based real time PCR assay for the rapid detection of channel catfish(Ictalurus punctatus) virus,the ORF8 gene of channel catfish virus was down-loaded from Genbank and the specific primers and probes were designed.The primers and probes as well as the reaction condition were optimized to improve the sensitivity and specificity of the assay.The specificity,sensitivity,reproducibility of the method were estimated and a standard curve was prepared.It was found that the specificity was high without any cross-reactions with kio herpesvirus,singapore grouper iridovirus,infectious spleen and kidney necrosis virus,sea bream iridovirus and other commonly encountered viruses from aquatic animals.A good linear correlation was demonstrated in the standard curve for the real-time PCR assay within the range from 3.3×10～3.3×107 copies.A minimum of 33 positive plasmids could be detected,indicating a good sensitivity of the assay.The coefficients of variation were 0.7% and 1.2% for the intra-assay and inter-assay tests respectively,indicating a good reliability.It took only 3 h to complete the whole course of reaction including extraction of viral DNA and the real-time PCR.The results demonstrate that real-time PCR method can be used as an effective detection and quantification method for CCV and it is amenable to high-throughout assay for its specificity,sensitivity and rapidity.
出处
《长江大学学报（自科版）（中旬）》
CAS
2008年第1期42-46,共5页
Journal of Yangtze University(Nature Science Edition)
基金
国家高技术研究发展计划(863计划)项目(2006AA100306)
国家质量监督检验检疫总局资助项目(2006IK003)
