Abstract
Getah virus (GETV), a mosquito-borne alphavirus, is an emerging animal pathogen causing outbreaks among racehorses and pigs. Early detection of the GETV infection is essential for timely implementation of disease prevention and control interventions. Thus, a rapid and accurate nucleic acid detection method for GETV is highly needed. Here, two TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays were developed. The qRT-PCR primers and TaqMan MGB probe were designed based on the conserved region of nsP1 and nsP2 genes of 23 GETV genome sequences retrieved from GenBank. Only the qRT-PCR assay using nsP2-specific primers and probe detected all two Malaysia GETV strains (MM2021 and B254) without cross-reacting with other closely related arboviruses. The qRT-PCR assay detected as few as 10 copies of GETV RNA, but its detection limit at the 95% probability level was 63.25 GETV genome copies (probit analysis, P ≤ 0.05). Further validation of the qRT-PCR assay using 16 spiked simulated clinical specimens showed 100% for both sensitivity and specificity. In conclusion, the qRT-PCR assay developed in this study is useful for rapid, sensitive and specific detection and quantification of GETV.
Highlights
Getah virus (GETV) is mosquito-borne virus belonging to the genus Alphavirus of the family Togaviridae
The in silico study revealed that both sets of GETV primers and probes showed mismatches with sequences of other alphaviruses including Sindbis virus, Ndumu virus, Chinkungunya virus, O’Nyong Nyong virus, Semliki Forest virus, Una virus, Mayaro virus, Ross River virus, Middelburg virus, Barmah Forest virus, Eastern equine encephalitis virus, Western equine encephalitis virus and Venezuelan equine encephalitis virus (Supplementary Fig. S2)
The findings showed that the assay using nsP2 primers and probe was better for the detection and quantification of GETV RNA as it could detect all the GETV strains used in this study without cross-reacting with other common arboviruses including Chikungunya virus (CHIKV), Sindbis virus (SINV), DENV-1, DENV-2, DENV-3, DENV-4, Zika virus (ZIKV) P6-740, ZIKV MR766, Japanese encephalitis virus (JEV), and LGT
Summary
Getah virus (GETV) is mosquito-borne virus belonging to the genus Alphavirus of the family Togaviridae. In 2017, the first GETV outbreak among pigs occurred in China resulting in hundreds deaths in the piglets[22]. Both horses and pigs act as an amplifying host of GETV as they developed high enough viremia titer to infect biting mosquitoes and maintain the GETV transmission cycle[18,23,24,25]. Virus isolation and serological tests, for instance serum neutralization test, are commonly performed for the diagnosis of GETV infection[17,22,26] In this study, we intended to develop a quantitative RT-PCR (qRT-PCR) assay for rapid detection and quantification of GETV
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