Quantitative detection method of Enterocytozoon hepatopenaei using TaqMan probe real-time PCR

Ya-Mei Liu; Liang Qiu; An-Zhi Sheng; Xiao-Yuan Wan; Dong-Yuan Cheng; Jie Huang

doi:10.1016/j.jip.2017.12.006

Quantitative detection method of Enterocytozoon hepatopenaei using TaqMan probe real-time PCR

J Invertebr Pathol. 2018 Jan:151:191-196. doi: 10.1016/j.jip.2017.12.006. Epub 2017 Dec 23.

Authors

Ya-Mei Liu¹, Liang Qiu¹, An-Zhi Sheng², Xiao-Yuan Wan², Dong-Yuan Cheng¹, 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: 29277659
DOI: 10.1016/j.jip.2017.12.006

Abstract

A TaqMan probe and a pair of specific primers were selected from the small subunit ribosomal DNA (SSU rDNA) sequence of Enterocytozoon hepatopenaei (EHP); this real-time PCR assay was developed and optimized. It showed a good linearity in detecting standards of EHP SSU rDNA fragments from 4 × 10² to 4 × 10⁸ copies/reaction using the established method. The detection limit of the qPCR method was as low as 4 × 10¹ copies per reaction, which was higher than the conventional PCR and SYBR Green I-based EHP qPCR reported. Using the qPCR assay, EHP was detected in four batches of slow-growing Penaeus vannamei specimens collected from Tianjin and Zhejiang Province in China was detected using qPCR. The results showed that all the hepatopancreas from the slow-growing P. vannamei specimens were detected as EHP-positive. EHP copies of hepatopancreas in some batches had a negative correlation with the body mass index (BMI) of shrimps; however, not all batches of specimens had this negative correlation between EHP copies of hepatopancreas and BMI. This qPCR technique is sensitive, specific and easy to perform (96 tests in <3 h), which provides technical support for the detection and prevention of EHP.

Keywords: Body mass index (BMI); Enterocytozoon hepatopenaei (EHP); TaqMan-based qPCR.

Publication types

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

MeSH terms

Animals
DNA, Fungal / analysis*
DNA, Ribosomal / analysis
Enterocytozoon*
Penaeidae / parasitology*
Real-Time Polymerase Chain Reaction / methods*

Substances

DNA, Fungal
DNA, Ribosomal