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.

Introduction: Retinitis pigmentosa (RP) is a group of hereditary retinal diseases in which photoreceptor cells degenerate. It is both clinically and genetically heterogenous. Using a two stage approach by combining linkage analysis with mutation detection, we have rapidly identified the gene locus and the mutation site of a Chinese Singaporean family with autosomal dominant RP. Materials and Methods: Three Chinese Singaporean families were tested. One family showed autosomal dominant inheritance pattern, while the other two could be recessive or sporadic. Twelve di-nucleotide markers tightly linked to 6 genes known to be responsible for either autosomal dominant or recessive RP were selected for linkage analysis. Cosegregation of marker and disease inheritance pattern permits identification of the target candidate gene. RFLP (restriction fragment length polymorphism) markers were added to confirm the linkage result prior to the detailed mutation detection study. Results: With this two-stage strategy, the autosomal dominant RP family showed the rhodopsin locus segregating concordantly with the disease. Mutation screening later identified a nonsense mutation 5261C>T in the last exon of rhodopsin gene. It predicted a Q344X changes at the C-terminus of the gene product, truncating it by 5 amino acids. Conclusion: This systematic approach facilitates molecular diagnosis of a genetically heterogenous disease like RP. This is the first report of an RP mutation in Singapore. This 5261C>T mutation has been reported in the Caucasian, but not the Chinese population. The relatively milder phenotype in this family showed similarity to the reported US family, indicating the correlation of mutation site to severity of disease regardless of ethnicity.