A TaqMan RT-qPCR assay for tilapia lake virus (TiLV) detection in tilapia

Waiyamitra, Pitchaporn; Tattiyapong, Puntanat; Sirikanchana, Kwanrawee; Mongkolsuk, Skorn; Nicholson, Pamela; Surachetpong, Win

doi:10.1016/j.aquaculture.2018.07.060

Cited by 49 publications

(38 citation statements)

References 23 publications

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“…Ancillary testing for TiLV and Francisella noatunensis subsp. orientalis , using protocols by Waiyamitra et al () and Soto et al (), respectively, was negative. Minimal inhibitory concentrations (MICs) were determined for the case isolates and select archived isolates using the Sensititre Vet Avian AVIAN1F Plate (Trek Diagnostic Systems), following the manufacturer's suggested protocol.…”

Section: Antimicrobial Minimum Inhibitory Concentrations (Mics) Againmentioning

confidence: 83%

Pathologic changes in cultured Nile tilapia (Oreochromis niloticus) associated with an outbreak of Edwardsiella anguillarum

Armwood

Camus

López-Porras

et al. 2019

Journal of Fish Diseases

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Section: Antimicrobial Minimum Inhibitory Concentrations (Mics) Againmentioning

confidence: 83%

Pathologic changes in cultured Nile tilapia (Oreochromis niloticus) associated with an outbreak of Edwardsiella anguillarum

Armwood

Camus

López-Porras

et al. 2019

Journal of Fish Diseases

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“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Taengphu

Sangsuriya

Phiwsaiya

et al. 2019

Preprint

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AbstractThe gene of RNA viruses, encoding RNA-directed RNA polymerase (RdRp) is relatively conserved due to its crucial function in viral genome replication and transcription making it a useful target for genetic diversity study and PCR detection. In this study, we investigated the genetic diversity of 21 tilapia lake virus (TiLV) genome segment 1 sequences predictively coding for RdRp subunit P1. Those sequences were obtained from infected fish samples collected in Ecuador, Israel, Peru, and Thailand between 2011 and 2019 (nine sequences from this study and 12 sequences from GenBank). Primers were then designed from the highly conserved regions among all 21 TiLV segment 1 sequences and used in semi-nested RT-PCR condition optimization. The result revealed that all 21 TiLV segment 1 sequences showed 95.00-99.94 and 99.00-100% nucleotide and amino acid sequence identity, respectively. These isolates were phylogenically clustered into three separate genetic clades, called i) Israeli-2011 clade (containing of TiLV isolates from Israel collected in 2011, Ecuador, and Peru isolates), ii) monophyletic Israel-2012 clade (containing only TiLV isolates collected from Israel in 2012), and iii) Thai clade (containing only sequences obtained from Thailand isolates). The newly established PCR protocol was 100 times more sensitive than our previous segment 3-based protocol when comparatively assayed with RNA extracted from infected fish. The assay was also shown to be specific when tested against negative control samples, i.e. RNA extracted from clinical healthy tilapia and from bacterial and viral pathogens (other than TiLV) commonly found in aquatic animals. Validation experiment with RNA extracted from naturally infected fish specimens collected in 2013-2019 yielded positive test results for all samples tested, confirming that our newly designed primers and detection protocol against TiLV segment 1, have a potential application for detection of all current genetic variants of TiLV.

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“…The melting curve analyses in the representative results shown from two laboratories did not reveal the presence of primer-dimers or other unwanted PCR products but if this is observed with other samples and/or experimental set-ups, then the assay should be re-optimized. More advanced qPCR technologies do not require such a melting curve step and indeed, since this methods paper was written, a TaqMan based TiLV RT-qPCR has been developed utilizing two primers and a probe making it highly TiLV specific 34 .…”

Section: Discussionmentioning

confidence: 99%

“…Such methods should allow early detection of infection before clinical signs develop and detection of low virus loads. To date, different PCR protocols including RT-PCR 14,32 , nested RT-PCR 18 , semi-nested RT-PCR 33 , and RT-qPCR 32,34 have been developed for the detection of TiLV in fish tissues. A comparison of RT-qPCR and virus isolation in susceptible cell lines for TiLV detection revealed that RT-qPCR was 1,000 times more sensitive than the virus isolation 32 .…”

Section: Introductionmentioning

confidence: 99%

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

Nicholson

Rawiwan

Surachetpong

2018

JoVE

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The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be used as part of surveillance programs, biosecurity measures and in TiLV basic research laboratories. The gold standard of virus diagnostics typically involves virus isolation followed by complementary techniques such as reverse-transcription polymerase chain reaction (RT-PCR) for further verification. This can be cumbersome, time-consuming and typically requires tissue samples heavily infected with virus. The use of RTquantitative (q)PCR in the detection of viruses is advantageous because of its quantitative nature, high sensitivity, specificity, scalability and its rapid time to result. Here, the entire method of PCR based approaches for TiLV detection is described, from tilapia organ sectioning, total ribonucleic acid (RNA) extraction using a guanidium thiocyanate-phenol-chloroform solution, RNA quantification, followed by a two-step PCR protocol entailing, complementary deoxyribonucleic acid (cDNA) synthesis and detection of TiLV by either conventional PCR or quantitative identification via qPCR using SYBR green I dye. Conventional PCR requires post-PCR steps and will simply inform about the presence of the virus. The latter approach will allow for absolute quantification of TiLV down to as little as 2 copies and thus is exceptionally useful for TiLV diagnosis in sub-clinical cases. A detailed description of the two PCR approaches, representative results from two laboratories and a thorough discussion of the critical parameters of both have been included to ensure that researchers and diagnosticians find their most suitable and applicable method of TiLV detection.

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A TaqMan RT-qPCR assay for tilapia lake virus (TiLV) detection in tilapia

Cited by 49 publications

References 23 publications

Pathologic changes in cultured Nile tilapia (Oreochromis niloticus) associated with an outbreak of Edwardsiella anguillarum

Pathologic changes in cultured Nile tilapia (Oreochromis niloticus) associated with an outbreak of Edwardsiella anguillarum

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

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

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