Efficiency, sensitivity and specificity of a quantitative real-time PCR assay for Tilapia Lake virus (TiLV)

Augustino Alfred Chengula, Kizito Kahoza Mugimba, Shlomit Tal, Roni Tadmor Levi, Saurabh Dubey, Stephen Mutoloki, Arnon Dishon, Lior David, Øystein Evensen, Hetron Mweemba Munang'andu*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Tilapia lake virus (TiLV) is an emerging viral pathogen of tilapiines worldwide in wild and farmed tilapia. TiLV is an orthomyxo-like, negative sense segmented RNA virus, belonging to genus Tilapinevirus, family Amnoonviridae. Here we developed a quantitative real-time PCR (qRT-PCR) assay testing primer sets targeting the 10 segments of TiLV. Sensitivity, specificity, efficiency and reproducibility of these assays were examined. Detection sensitivity was equivalent to 2 TCID50/ml when tested on supernatants from cell culture-grown TiLV. Specificity tests showed that all primer sets amplified their respective TiLV segments, and standard curves showed linear correlation of R2 > 0.998 and amplification efficiencies between 93 % and 98 %. Intra- and inter-assay coefficients of variation (CV %) were in the range of 0.0 %− 2.6 % and 0.0 %− 5.9 %, respectively. Sensitivity tests showed that primer sets targeting segments 1, 2, 3 and 4 had the highest detection sensitivities (100.301 TCID50/ml). The qRT-PCR used for detection of viral genome in TiLV infected organs gave virus titers equivalent to 3.80 log10, 3.94 log10 and 3.52 log10 TCID50/ml for brain, kidney and liver tissues, respectively as calculated on the basis of Ct values. These findings suggest that primer optimization for qPCR should not only focus on attaining high amplification efficiency but also sensitivity comparison of primer sets targeting different viral segments in order to develop a method with the highest sensitivity.

Original languageAmerican English
Article number114567
JournalJournal of Virological Methods
Volume307
DOIs
StatePublished - Sep 2022

Bibliographical note

Publisher Copyright:
© 2022 Elsevier B.V.

Keywords

  • Amplification efficiency
  • Polymerase chain reaction
  • Quantitative real-time PCR
  • Sensitivity
  • Specificity
  • Tilapia Lake virus

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