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Development of a simple and rapid reverse transcription–loopmediated isothermal amplification (RT‐LAMP) assay for sensitive detection of tilapia lake virus
Author(s) -
Yin Jiyuan,
Wang Qing,
Wang Yingying,
Li Yingying,
Zeng Weiwei,
Wu Jiexing,
Ren Yan,
Tang Yafang,
Gao Caixia,
Hu Huzi,
Bergmann Sven M.
Publication year - 2019
Publication title -
journal of fish diseases
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.819
H-Index - 85
eISSN - 1365-2761
pISSN - 0140-7775
DOI - 10.1111/jfd.12983
Subject(s) - loop mediated isothermal amplification , biology , reverse transcription loop mediated isothermal amplification , tilapia , virus , microbiology and biotechnology , virology , sybr green i , reverse transcription polymerase chain reaction , polymerase chain reaction , reverse transcriptase , gene , fish <actinopterygii> , dna , gene expression , genetics , fishery
Recently, substantial mortality of farmed and wild tilapia caused by tilapia lake virus (TiLV) infection has been observed worldwide. However, sensitive and reliable diagnostic method is limited. A reverse transcription–loopmediated isothermal amplification (RT‐LAMP) assay has been applied for the detection of TiLV nucleotide sequence. Six primers targeting two locations on the target gene based on a highly conserved sequence in the segment 1 (S1) region of the TiLV genome have been designed. The optimized RT‐LAMP reaction was maintained at the isothermal condition of 63°C for 45 min. And the amplifications could be verified by turbidity or a colour change with the addition of SYBR Green I. Subsequently, RT‐LAMP products could be observed by a ladder pattern following gel electrophoresis. The species‐specific assay showed that the method was sensitive enough to detect as low as 1.6 copies of viral particle, and the assay was highly specific because no cross‐reactivity was observed with other pathogens, and had a diagnostic sensitivity and specificity of 100% when TiLV‐positive samples and non‐target virus were tested. In summary, all the results demonstrate that this RT‐LAMP is a rapid, effective and sensitive method for TiLV detection in tilapia aquaculture.