Comparison Of Reverse Transcription Polymerase Chain Reaction Methods For The Detection Of Tilapia Lake Virus

Tilapia Lake Virus disease (TiLVD) is an emerging viral disease associated with high morbidity and mortality in tilapia worldwide. Therefore, rapid identification and quantification of TiLV is crucial for implementing appropriate control measures to reduce the spread of the disease. Here, we compared four reverse transcription polymerase chain reaction (RT-PCR) and two RT-quantitative PCR (RT-qPCR) methods to detect TiLV. Performance characteristics for each assay were evaluated using a serial dilution of TiLV-infected E-11 cells, and testing tilapia tissues with known infection status. Among these molecular methods, the SYBR RT-qPCR was the most sensitive assay followed by semi-nested RT-PCR(1), TaqMan RT-qPCR, nested RT-PCR, semi-nested RT-PCR(2), and RT-PCR. Further validation of these assays using 40 laboratory challenged fish tissues; 20 TiLV positive samples and 20 negative samples revealed that all six PCR methods could detect the virus with different sensitivity. In particular, the SYBR RT-qPCR, TaqMan RT-qPCR, nested RT-PCR, and semi-nested RT-PCR(1) methods showed 100% accuracy to detect the presence of TiLV in samples. In contrast, the semi-nested RT-PCR(2) and RT-PCR methods exhibited some false-negative results at 10% and 35%, respectively. No false-positive TiLV results were observed for all six RT-PCR protocols. Collectively, these findings support the decision for selecting an appropriate diagnostic assay for TiLV.