Development of a simple and rapid reverse transcription–loopmediated isothermal amplification (RT‐LAMP) assay for sensitive detection of tilapia lake virus

Abstract

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.

Tilapia lake virus: The story so far

“…The application of RT‐PCR is not particularly suitable for rapid and on‐site disease diagnosis. Therefore, two RT‐loop‐Mediated isothermal amplification (RT‐LAMP) assays based on the colorimetric changes which offer rapid, cost‐effective and easy to interpret result have been recently developed (Phusantisampan, Tattiyapong, Mutrakulcharoen, Sriariyanun, & Surachetpong, 2019; Yin et al., 2019). Both RT‐LAMP assays showed sensitivities of 80.95% and 100% for TiLV, respectively (Phusantisampan et al., 2019; Yin et al., 2019).…”

Genetic diversity of tilapia lake virus genome segment 1 from 2011 to 2019 and a newly validated semi-nested RT-PCR method

“…Rapid and accurate detection of the virus is crucial for selection of TiLV-free fish broodstock in view of the vertical transmission possibilities of the virus from parents to offspring (Yamkasem et al 2019; Dong et al 2020) and to prevent disease spread through movement of live fish for aquaculture (Dong et al 2017a; Jansen et al 2018). Following the characterization of the TiLV genomes (Eyngor et al 2014; Bacharach et al 2016), several PCR methods have been published, including RT-PCR (Eyngor et al 2014), nested RT-PCR (Kembou Tsofack et al 2017), semi-nested RT-PCR (Dong et al 2017b), RT-qPCR (Kembou Tsofack et al 2017; Tattiyapong et al2018; Waiyamitra et al 2018), and RT-LAMP (Phusantisampan et al 2019; Yin et al 2019). Most of the aforementioned methods used genome segment 3 of TiLV as the target for primer design.…”

Detection of melon necrotic spot virus by one-step reverse transcription loop-mediated isothermal amplification assay

“…Numerous studies have confirmed that the RT-LAMP method has greater sensitivity than either standard RT-PCR or nested RT-PCR [20,22,31]. The visual assessment of the RT-LAMP assay can be independent of expensive equipment such as a PCR thermocycler or a metal thermostat-controlled heater.…”

“…Conventional detection of MNSV is performed by enzyme-linked immunosorbent assay (ELISA) or reverse transcription-polymerase chain reaction (RT-PCR) [18], but these methods have the disadvantages of taking a long time to complete, and of requiring the availability of specialized equipment and expensive consumables. Recent research has used the reverse transcription-loop-mediated isothermal amplification (RT-LAMP) to detect plant [19,20], animal [21,22] and human viruses [23,24]. This method has been shown to exhibit high sensitivity, reliability and rapid completion time; furthermore, it requires cheaper consumables and simpler equipment than the conventional RT-PCR detection method.…”