Intensification of shrimp farming has led to the development of a number of diseases which result in enormous loss in shrimp production within the farm with subsequent significant economic impact to the farmer. Existing diagnostic tests for detecting shrimp pathogens are limited to the detection of one pathogen at a time. It is highly challenging to detect these pathogens at times of co-infections.
The present study reports a simple and label-free diagnostic method for simultaneous detection of four different shrimp pathogens namely White Spot Syndrome Virus (WSSV), Monodon Baculovirus (MBV), Necrotizing Hepatopancreatitis Bacterium (NHPB) and Infectious Hypodermal & Haemopoietic Necrosis Virus (IHHNV). This method is based on multiplex PCR and detection by high resolution melting (HRM) profile. HRM analysis involves PCR amplification of the region of interest in the presence of double stranded DNA binding dye followed by melting at high resolution. Based on this principle, we designed primers in such way that the melting point (Tm) of PCR products differ at least 1.5°C so the peaks of four different PCR products can be distinguished easily. Besides, varying concentrations and ratios of primer pairs and salt are crucial to the successful amplification of all the four targets equally in a multiplex PCR reaction. The target region of the DNA and primers for the detection of WSSV (Durand & Lightner, 2002), IHHNV (Dhar et al., 2009), and NHPB (Nunan et al., 2008) and MBV (Tang et al., 2011) were essentially similar to the one described in the published reports with slight modification to meet the required Tm.
A 4-plex assay for WSSV, IHHNV, MBV and NHPB detection was developed using the plasmid templates and then evaluated with genomic DNA and field samples. The WSSV, MBV, IHHNV and NHPB target DNA were represented by 66, 120, 121 and 163 base pair (bp) PCR products respectively. The PCR products were also analyzed by 2% agarose gel and an intense band could be observed in the range of 150bp. HRM profile of the PCR products of MBV, WSSV, IHHNV and NHPB were analyzed from both individual (monoplex) and 4-plex reactions. The results indicated that the Tm of MBV, WSSV, IHHNV and NHPB were 74.9, 79.0, 81.6 and 84.5°C respectively. There is no significant difference in Tm of these PCR products compared to the individual (monoplex) and 4-plex HRM reactions.
Based on the results, it is concluded that the HRM assay is rapid, sensitive and allows simultaneous detection of multiple pathogens in shrimp with mixed or co-infection. This diagnostic method has great potential in shrimp industry and regulatory bodies in bringing down the cost, time and man-power besides helping to control the disease outbreak.