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

doi:10.1016/j.jviromet.2022.114567

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

Department of Animal Sciences

Research output: Contribution to journal › Article › peer-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 (10^0.301 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 language	American English
Article number	114567
Journal	Journal of Virological Methods
Volume	307
DOIs	https://doi.org/10.1016/j.jviromet.2022.114567
State	Published - Sep 2022

Bibliographical note

Funding Information:
This work was supported by the TRAHESA project responsible for capacity building through training and research in aquatic and environmental health in Eastern and Southern Africa, financed by the Norwegian Development Agency for Development Cooperation (NORAD) [grant numbers QZA-0485 RAF-19/0018 ]; and partly financed by the Research Council of Norway [grant number 283566 BIOAQUA ].

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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10.1016/j.jviromet.2022.114567

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

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.",

keywords = "Amplification efficiency, Polymerase chain reaction, Quantitative real-time PCR, Sensitivity, Specificity, Tilapia Lake virus",

author = "Chengula, {Augustino Alfred} and Mugimba, {Kizito Kahoza} and Shlomit Tal and Levi, {Roni Tadmor} and Saurabh Dubey and Stephen Mutoloki and Arnon Dishon and Lior David and {\O}ystein Evensen and Munang'andu, {Hetron Mweemba}",

note = "Funding Information: This work was supported by the TRAHESA project responsible for capacity building through training and research in aquatic and environmental health in Eastern and Southern Africa, financed by the Norwegian Development Agency for Development Cooperation (NORAD) [grant numbers QZA-0485 RAF-19/0018 ]; and partly financed by the Research Council of Norway [grant number 283566 BIOAQUA ]. Publisher Copyright: {\textcopyright} 2022 Elsevier B.V.",

year = "2022",

month = sep,

doi = "10.1016/j.jviromet.2022.114567",

language = "American English",

volume = "307",

journal = "Journal of Virological Methods",

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T1 - Efficiency, sensitivity and specificity of a quantitative real-time PCR assay for Tilapia Lake virus (TiLV)

AU - Chengula, Augustino Alfred

AU - Mugimba, Kizito Kahoza

AU - Tal, Shlomit

AU - Levi, Roni Tadmor

AU - Dubey, Saurabh

AU - Mutoloki, Stephen

AU - Dishon, Arnon

AU - David, Lior

AU - Evensen, Øystein

AU - Munang'andu, Hetron Mweemba

N1 - Funding Information: This work was supported by the TRAHESA project responsible for capacity building through training and research in aquatic and environmental health in Eastern and Southern Africa, financed by the Norwegian Development Agency for Development Cooperation (NORAD) [grant numbers QZA-0485 RAF-19/0018 ]; and partly financed by the Research Council of Norway [grant number 283566 BIOAQUA ]. Publisher Copyright: © 2022 Elsevier B.V.

PY - 2022/9

Y1 - 2022/9

N2 - 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.

AB - 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.

KW - Amplification efficiency

KW - Polymerase chain reaction

KW - Quantitative real-time PCR

KW - Sensitivity

KW - Specificity

KW - Tilapia Lake virus

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DO - 10.1016/j.jviromet.2022.114567

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AN - SCOPUS:85132770024

SN - 0166-0934

VL - 307

JO - Journal of Virological Methods

JF - Journal of Virological Methods

M1 - 114567

ER -

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

Abstract

Bibliographical note

Keywords

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