Taqman-MGB探针荧光定量PCR方法检测对虾传染性皮下及造血组织坏死病毒的研究  

Quantitative detection of infectious hypodermal and hematopoietic necrosis virus using Taqman-MGB probe

作　　者：徐丽美[1] 李福英[1] 

机构地区：[1]国家海洋局第三海洋研究所、国家海洋局海洋生物遗传资源重点实验室,福建厦门361005

出　　处：《应用海洋学学报》2013年第1期133-137,共5页Journal of Applied Oceanography

基　　金：国家公益性行业(农业)科研专项资助项目(201103034);国家公益性行业(海洋)科研专项资助项目(200905020)

摘　　要：本文根据GenBank中对虾传染性皮下及造血组织坏死病毒(IHHNV)基因组上的保守序列,采用Taqman-MGB荧光定量PCR方法,设计了一组特异性引物和探针.利用阳性标准模板进行了定量PCR条件的优化研究.结果显示,在101～108拷贝范围内标准曲线的线性关系为R=0.999 4,检测灵敏度达到10拷贝,重复检测的变异系数均小于2%.利用上述建立的方法和条件,测定39份不同来源的对虾样品中IHHNV,检出率为49%.因此所建立的荧光定量PCR具有较高的灵敏度、特异性和重复性,可作为IHHNV快速的定量检测方法.Infectious hypodermal and hematopoietic necrosis virus （IHHNV） is one of important pathogen in shrimp that causes negative effects on shrimp aquaculture and the disease agent is listed by the OIE （World Organization for Animal Health）. Based on the conserved sequence in the IHHNV genome in GenBank, a set of specific primers and Taqman-MGB probe was designed and combined with positive standard template to optimize quantitative PCR conditions. The results showed that a strong linear relationship （R =0. 999 4） ranging from 101 to l0s copies was obtained and its detection limit was 10 copies, The coefficients of variation for repeated measurements were less than 2%. The established assay was then used to quantify IHHNV in 39 shrimp specimens from different sources. The IHHNV detection rate was 49%. Therefore, the qPCR method for rapid quantitative detection of IHHNV has a high sensitivity, specificity and reproducibility.

关 键 词：海洋生物学 传染性皮下及造血组织坏死病毒(IHHNV) 荧光定量PCR TAQMAN-MGB探针 

分 类 号：P735[天文地球—海洋生物学]