Validation of qPCR from a crude extract for the rapid detection of white spot syndrome virus

Abstract

White spot disease (WSD) has posed a serious threat to the China and the global shrimp aquaculture. In order to diagnose white spot syndrome virus (WSSV) early and prevent the spread and outbreak of WSD, it is necessary to establish a highly sensitive WSSV diagnosis method suitable for shrimp farming sites. In this study, a pre-amplification qPCR assay from the crude extract of samples heated lysis was established, which was further compared with the universal qPCR assay to verify the shrimp samples. The limit of detection (LOD) of pre-amplification qPCR assay and universal qPCR assay was 2.80 copies and 20.57 copies per reaction at 95% CI, respectively. It had good WSSV specificity and did not show cross-detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV), hepatopancreatic parvovirus (HPV), Enterocytozoon hepatopenaei (EHP), acute hepatopancreas necrosis disease (AHPND), necrotizing hepatopancreatitis bacteria (NHPB), and decapod iridescent virus 1 (DIV1). A total of 36 shrimp samples were detected as WSSV DNA positive by pre-amplification qPCR with crude extract from samples heated lysis and universal qPCR with DNA extraction. The diagnostic sensitivity and specificity were 97.22% (85.5 ~ 99.9%, 95% CI) and 100% (81.5 ~ 100%, 95% CI), respectively. The agreement Kappa value was 0.959 (0.879 ~ 1, 95% CI), and the analysis results were basically consistent. Eliminating the tedious steps of extracting DNA and using pre-amplified qPCR to detect WSSV in shrimp, it is a good choice for aquaculture farms. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s10499-022-00920-9.

Living Magnetotactic Microrobots Based on Bacteria with a Surface-Displayed CRISPR/Cas12a System for Penaeus Viruses Detection

“…The significant new viruses decapod iridescent virus 1 (DIV1) and white spot syndrome virus (WSSV), which are detrimental to shrimp aquaculture, have greatly harmed and seriously damaged the shrimp farming business in China and around the world. , There are no specific medications available as of now. So, one of the most important steps in preventing the propagation and outbreak of the virus is quick and precise surveillance at the shrimp breeding location. , DIV1 and WSSV can be identified by their characteristic markers, the ATPase gene of DIV1 (GenBank: KY681040.1) and the VP66 gene of WSSV (GenBank: MN481520.1). , Gene sequences were used to design crRNA-specific binding sites and isothermal amplification primers (Figure S6).…”

“…To verify the detection limits and sensitivity of bacterial microrobots, gene fragments of DIV1 (GenBank: KY681040.1) and WSSV (GenBank: MN481520.1) were inserted into the commercial plasmid backbone (pET28b), and the viral copy number was calculated based on the plasmid concentration versus the relative molecular mass. , Isothermal amplification was performed after gradient dilution of the plasmid, and the RPA kit (S001ZC, Hangzhou ZC Bio-Sci & Tech Co. Ltd.) was used for isothermal amplification. Amplicons were coincubated with bacterial microrobots for 0.5 h at 37 °C in assay buffer (20 mM HEPES, 10 mM MgCl 2 , 150 mM KCl, and 0.5 mM TCEP).…”

“…The significant new viruses decapod iridescent virus 1 (DIV1) and white spot syndrome virus (WSSV), which are detrimental to shrimp aquaculture, have greatly harmed and seriously damaged the shrimp farming business in China and around the world. 24,32 There are no specific medications available as of now. So, one of the most important steps in preventing the propagation and outbreak of the virus is quick and precise surveillance at the shrimp breeding location.…”