Establishment and Application of MCRV Detection Method for Larvae of Mud Crab, Scylla paramamosain

Abstract

Mud crab reovirus (MCRV) present in Scylla paramamosain larvae poses notable risks to the late stage of mud crab aquaculture. The cultivation of MCRV-free larvae and prevention of virus transmission at its source have become critical issues that must be urgently addressed in mud crab aquaculture. Owing to the low viral load in fertilized eggs and larvae of mud crabs, we attempted to establish a more sensitive detection method for MCRV. Most of the current MCRV detection methods are based on the VP1 and VP6 genes of MCRV, which limit the sensitivity of detection methods owing to the low expression of these two genes. Our previous studies found that detection sensitivity was significantly improved by using VP11 as the target gene because VP11 is the highest expression gene of MCRV in mud crabs. In addition, compared with SYBR Green qRT-PCR, TaqMan-MGB qRT-PCR typically has higher sensitivity in theory. Therefore, in this study, we selected the highly expressed VP11 of MCRV as the target gene, designed specific primers and TaqMan-MGB probes, and established a qRT-PCR detection method suitable for MCRV in mud crab larvae by optimizing the reaction system and procedure, using standard plasmid and RNA as the templates. The final reaction components were as follows: 10 μL Premix Ex Taq (Probe qPCR), 0.4 μL VP11-F (10 μmol/L), 0.4 μL VP11-R (10 μmol/L), 0.4 μL VP11-P (10 μmol/L), 0.4 μL ROX, 2 μL DNA template, and 6.8 μL ddH2O. The final procedure was as follows: Initial denaturation at 95 ℃ for 3 min, followed by 40 cycles of 95 ℃ for 10 s and 60 ℃ for 30 s. The results showed a good linear relationship between the log value (starting quality, Sq) and number of reaction cycles within the range of 1×108 – 1×101 copies/µL of standard plasmids (or dsRNA). Standard plasmids or dsRNA can be accurately quantified with a minimum of 10 copies, and the minimum theoretical detection limit is 2.5 copies/reaction of standard plasmids. The coefficients of variation of this detection method for plasmid and dsRNA samples were 1.4% and 0.98%, respectively, showing good repeatability and stability. When MCRV, MCDV, WSSV, DIV1, EHP, and Vibrio parahaemolyticus nucleic acid samples were used as templates for MCRV detection, no specific amplification was observed except for MCRV samples, indicating that this detection method is highly specific for MCRV. To further validate the practicality and sensitivity of TaqMan-MGB qRT-PCR, 14 batches of mud crab larvae with suspected MCRV infection were detected using this method, and SYBR Green qRT-PCR method was also used to analyze the samples simultaneously. The MCRV detection rate of the former was 78.57%, and the latter was only 57.14%, indicating that TaqMan-MGB qRT-PCR has a higher sensitivity than the SYBR Green method. These findings indicate that the newly established detection method has strong specificity and stability, as well as higher sensitivity. This detection method can be applied to screening MCRV in mud crab larvae at different developmental stages and provide technical support for the production of MCRV-free mud crab seedlings.

Keywords

• scylla paramamosain
• mcrv
• detection method
• taqman qrt-pcr
• larvae