Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of white spot syndrome virus and infectious hypodermal and hematopoietic necrosis virus in penaeid shrimp

Abstract

A multiplex LAMP (mLAMP) assay was developed for the simultaneous detection of two penaeid shrimp viruses, namely, white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV), which are the major viral pathogens of penaeid shrimp worldwide. Two sets of the LAMP primers were designed for VP28 gene of WSSV and non-structural protein 1 (NS1) gene of IHHNV, in which a restriction enzyme cleavage site was inserted into two pairs of species-specific primers, to construct the mLAMP assay by combining these two sets totaling eight primers. The mLAMP method was distinguishable between WSSV and IHHNV due to the subsequent restriction enzyme analysis. The sensitivities of the mLAMP method were 102 and 103 times higher on the detection limits for WSSV and IHHNV, respectively, than those of the nested-PCR (nPCR) and classical PCR methods. No positive results were displayed when nucleic acids from other shrimp pathogen DNAs were used as mLAMP templates. Uracil-N-glycosylase (UNG) was used to prevent carry-over contamination of mLAMP products. In addition to its high specificity and sensitivity, the developed multiplex LAMP assay offers an efficient, convenient and rapid tool for screening penaeid shrimp viruses since both WSSV and IHHNV can be diagnosed in a single reaction, and will be useful for the control of these viruses in shrimp.