Detection and quantification of shrimp hemocyte iridescent virus by TaqMan probe based real-time PCR

Liang Qiu; Meng-Meng Chen; Xiao-Yuan Wan; Qing-Li Zhang; Chen Li; Xuan Dong; Bing Yang; Jie Huang

doi:10.1016/j.jip.2018.04.005

Detection and quantification of shrimp hemocyte iridescent virus by TaqMan probe based real-time PCR

J Invertebr Pathol. 2018 May:154:95-101. doi: 10.1016/j.jip.2018.04.005. Epub 2018 Apr 13.

Authors

Liang Qiu¹, Meng-Meng Chen¹, Xiao-Yuan Wan², Qing-Li Zhang¹, Chen Li², Xuan Dong², Bing Yang², Jie Huang³

Affiliations

¹ Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; Shanghai Ocean University, Shanghai 201306, China.
² Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China.
³ Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; Shanghai Ocean University, Shanghai 201306, China. Electronic address: huangjie@ysfri.ac.cn.

PMID: 29660347
DOI: 10.1016/j.jip.2018.04.005

Abstract

Shrimp hemocyte iridescent virus (SHIV) is a recently reported shrimp virus, which threats the cultured white-leg shrimp Litopenaeus vannamei and can cause huge economic loss in shrimp farming industry in China. A quantitative real time polymerase chain reactio (qPCR) assay was developed using a TaqMan probe to detect and quantify SHIV. A pair of qPCR primers, which amplify a 188 bp DNA fragment, and a TaqMan probe were selected from ATPase gene (ORF114R) of the SHIV genome. The primers and TaqMan probe used in this assay were shown to be specific for SHIV and did not react with White spot syndrome virus (WSSV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Hepatopancreatic parvovirus (HPV), Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VP_AHPND), and Enterocytozoon hepatopenaei (EHP), or healthy shrimp DNA. The detection limit of the qPCR method was as low as 4 copies per reaction. The diagnostic sensitivity and the diagnostic specificity of the qPCR compared with nested PCR were 95.3% and 99.2%, respectively. The resulting standard curves showed high correlation coefficient values (R² = 0.998) in the range of 4 × 10⁹ to 4 × 10⁰ DNA copies/reaction. Test of farm samples showed that SHIV was detected in L. vannamei, Fenneropenaeus chinensis and Macrobrachium rosenbergii contained SHIV ranged from 1.21E+02 to 7.95E+07 copies (μg DNA)^-1. Quantitative detection of different tissues in artificial infected shrimp showed that haemolymph contained the highest SHIV load and muscle contained lowest SHIV load.

Keywords: Hemolymph; Litopenaeus vannamei; Quantitative real time PCR (qPCR); SHIV; Viral load.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Iridoviridae / genetics*
Iridoviridae / isolation & purification
Penaeidae / virology*
Real-Time Polymerase Chain Reaction
Sequence Analysis