Duplex droplet digital PCR method for the detection of Enterocytozoon hepatopenaei and Vibrio parahaemolyticus acute hepatopancreatic necrosis disease.

Enterocytozoon hepatopenaei (EHP) and Vibrio parahaemolyticus acute hepatopancreatic necrosis disease (VPAHPND) are two of the diseases that have frequently infected farmed shrimp in recent years, causing great economic losses to the shrimp industry worldwide. In this study, we established a sensitive and accurate duplex droplet digital PCR (ddPCR) method that can simultaneously detect and quantify the two pathogens simultaneously. The results showed that the ddPCR methods could detect EHP and VPAHPND specifically. The sensitivity levels of ddPCR for EHP and VPAHPND were 2.3 copies/µl and 4.6 copies/µl, respectively, which were 10-fold higher than the sensitivity of the qPCR assay and showed good reproducibility. Twenty-six suspected diseased shrimp samples were used for practical determination. For EHP, the detection rates of ddPCR and qPCR were 53.84% and 42.31%, respectively; for VPAHPND, the detection rates of ddPCR and qPCR were both 23.08%. The results indicated that the ddPCR method shows superiority for detection in samples with low viral loads, which will facilitate monitoring of the source and transmission of EHP and VPAHPND and will help control shrimp epidemic disease.

Development and comparison of qPCR and qLAMP for rapid detection of the decapod iridescent virus 1 (DIV1).

Decapod iridescent virus 1 (DIV1) is a new virus discovered in recent years that infects farmed shrimp. DIV1 is highly infectious and causes substantial economic loss to the aquaculture industry of China. To prevent and control the spread and outbreak of DIV1 in a timely manner, it is necessary to establish an efficient method for DIV1 diagnosis. In this study, quantitative real-time polymerase chain reaction (qPCR) and quantitative real-time loop-mediated isothermal amplification (qLAMP) detection methods were established based on the specific sequence of the viral ATPase gene. The results indicated that the minimum detection limits of qPCR and qLAMP were 1.9 × 101 copies/µL and 1.9 × 102 copies/µL, respectively; the designed primer had good specificity for DIV1 and did not react with 13 other viruses, including white spot syndrome virus (WSSV), Enterocytozoon hepatopenaei (EHP), acute hepatopancreatic necrosis disease (AHPND), infectious hypodermal and haematopoietic necrosis virus (IHHNV), etc. A total of 43 clinical samples suspected of DIV1 infection were diagnosed by qPCR and qLAMP. Our qPCR demonstrated results consistent with a qPCR assay published previously, and the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of qLAMP were 85.71% and 100%, respectively. This result indicates that qPCR and qLAMP have good accuracy in the detection of DIVI in clinical samples. As established in this study, qPCR and qLAMP combined with a comprehensive comparative analysis can provide effective new solutions for the detection of DIV1.