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

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.