荧光定量PCR与LAMP检测IHHNV的特异性和灵敏性比较

COMPARATIVE STUDY OF SPECIFICITY AND SENSITIVITY FOR IHHNV DETECTION BETWEEN REAL-TIME PCR AND LAMP METHODS

  • 摘要: 传染性皮下及造血器官坏死病毒(IHHNV)是世界各地养殖对虾的重要病毒性病原之一,给对虾养殖业造成严重经济损失。研究建立了检测IHHNV的荧光定量PCR和环介导等温核酸扩增(Loop-mediated isothermal amplification,LAMP)两种技术,并对它们的特异性和灵敏性进行了比较。结果显示,所建立的荧光定量PCR检测IHHNV的方法最低检测限度为6个DNA拷贝/反应,在待扩增DNA浓度为6.038×104-6.038×109cps/mL范围时,模板浓度与循环阈值Ct之间的相关性良好,决定系数r2为0.99521;对5份白斑综合症病毒基因组DNA和10份健康对虾基因组DNA样品进行荧光定量PCR检测,结果都为阴性;这说明荧光定量PCR检测IHHNV方法具有灵敏度高、特异性高和精确性高等优点。同样,所建立的LAMP检测IHHNV的方法在60min反应时间内也可检测到最低为6个拷贝的DNA模板,反应产物加入荧光染料SYBR Green I后反应液呈现明显的亮绿色,且特异的检测IHHNV DNA模板;这说明所建立的LAMP检测IHHNV的方法具有荧光定量PCR方法相当的灵敏度、特异性和精确性。考虑到LAMP检测方法操作更为简单、方便,而且不需要昂贵的仪器,LAMP检测IHHNV的方法更适合于对虾养殖现场检测的推广使用。

     

    Abstract: Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the most important pathogens infecting penaeid shrimp and causes huge economic losses in the shrimp culture industry worldwide. Because no commercial vaccine is yet available, the most effective way of containing the disease is by the routine screening of juveniles and adults for the presence or absence of the virus. Therefore, our goal is to establish a simple and rapid examination system for infectious hypodermal and hematopoietic necrosis virus in places such as shrimp ponds. Two detection methods, real-time PCR and loop-mediated isothermal amplification (LAMP), were developed for IHHNV diagnosis, and then their specificity and sensitivity were compared in the study. Using the real-time PCR method, the assay had a detection limit of six copies of DNA template of IHHNV, and had a correlation coefficient of 0.99521 between template concentration and threshold cycle value at the template concentration of 6.038×104 to 6.038×109cps/mL. Furthermore, the approach had no signal response to genomic DNA of white spot syndrome virus and shrimp. The result revealed that the detection method had high specificity and sensitivity for IHHNV detection. However, the costly real-time thermal cycler and technically demanding were deemed to be not appropriate for IHHNV detection in field conditions. LAMP was a novel, sensitive and rapid detection technique and could be applied for disease diagnosis in aquaculture. Here, a set of four primers was designed using PrimerExplorer V4 software by targeting the IHHNV genome DNA, and used to develop the LAMP method for IHHNV detection. Using the detection system, target DNA was amplified and visualized on agarose gel within 60min under isothermal condition at 64℃. Also, the LAMP amplicon was observed directly in the reaction tube by addition of SYBR Green I for a naked-eye inspection. Sensitivity assay showed that the method also had a detection limit of six copies of DNA template of IHHNV. Moreover, genomic DNA of white spot syndrome virus and shrimp were unable to be detected within 60min using the LAMP method. Overall, these data revealed that the LAMP method had an equivalent to the real-time PCR method in specificity and sensitivity for IHHNV detection. Considering that the LAMP method had great advantage in its performance and low cost, this technique was more suitable for IHHNV detection in field conditions. Therefore, the LAMP method could be applied routinely to check the shrimp in hatcheries and growout ponds, so that virus carrying shrimp could be found during the early infection stages and counter measures could be devised before the infection becomes epizootic.

     

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