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Detection and Quantification of Chikungunya Virus by Real-Time RT-PCR Assay

Wang SM, Ali UH, Sekaran SD, Thayan R

Methods Mol Biol, 2016;1426:105-17.
PMID: 27233265 DOI: 10.1007/978-1-4939-3618-2_10

Real-time PCR assay has many advantages over conventional PCR methods, including rapidity, quantitative measurement, low risk of contamination, high sensitivity, high specificity, and ease of standardization (Mackay et al., Nucleic Acids Res 30:1292-1305, 2002). The real-time PCR system relies upon the measurement of a fluorescent reporter during PCR, in which the amount of emitted fluorescence is directly proportional to the amount of the PCR product in a reaction (Gibsons et al., Genome Res 6:995-1001, 1996). Here, we describe the use of SYBR Green I-based and TaqMan(®) real-time reverse transcription polymerase chain reaction (RT-PCR) for the detection and quantification of Chikungunya virus (CHIKV).

Matched MeSH terms: Haplorhini/virology
Fulltext Mass spectrometry-based identification and whole-genome characterisation of the first pteropine orthoreovirus isolated from monkey faeces in Thailand

Kosoltanapiwat N, Reamtong O, Okabayashi T, Ampawong S, Rungruengkitkun A, Thiangtrongjit T, et al.

BMC Microbiol, 2018 10 17;18(1):135.
PMID: 30332986 DOI: 10.1186/s12866-018-1302-9

BACKGROUND: The pteropine orthoreovirus (PRV) was isolated from monkey (Macaca fascicularis) faecal samples collected from human-inhabited areas in Lopburi Province, Thailand. These samples were initially obtained to survey for the presence of hepatitis E virus (HEV).
RESULTS: Two virus isolates were retrieved by virus culture of 55 monkey faecal samples. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was successfully used to identify the viruses as the segmented dsRNA orthoreovirus. Phylogenetic analysis of the Lopburi orthoreovirus whole-genomes revealed relationships with the well-characterised PRVs Pulau (segment L1), Cangyuan (segments L2, M3 and S3), Melaka (segments L3 and M2), Kampar (segments M1 and S2) and Sikamat (segments S1 and S4) of Southeast Asia and China with nucleotide sequence identities of 93.5-98.9%. RT-PCR showed that PRV was detected in 10.9% (6/55) and HEV was detected in 25.5% (14/55) of the monkey faecal samples.
CONCLUSIONS: PRV was isolated from monkey faeces for the first time in Thailand via viral culture and LC-MS/MS. The genetic diversity of the virus genome segments suggested a re-assortment within the PRV species group. The overall findings emphasise that monkey faeces can be sources of zoonotic viruses, including PRV and HEV, and suggest the need for active virus surveillance in areas of human and monkey co-habitation to prevent and control emerging zoonotic diseases in the future.

Matched MeSH terms: Haplorhini/virology*
Fulltext Chikungunya virus in macaques, Malaysia

Sam IC, Chua CL, Rovie-Ryan JJ, Fu JY, Tong C, Sitam FT, et al.

Emerg Infect Dis, 2015 Sep;21(9):1683-5.
PMID: 26291585 DOI: 10.3201/eid2109.150439
Matched MeSH terms: Haplorhini/virology
Fulltext Development and evaluation of a one-step SYBR-Green I-based real-time RT-PCR assay for the detection and quantification of Chikungunya virus in human, monkey and mosquito samples

Ummul Haninah A, Vasan SS, Ravindran T, Chandru A, Lee HL, Shamala Devi S

Trop Biomed, 2010 Dec;27(3):611-23.
PMID: 21399603 MyJurnal

This paper reports the development of a one-step SYBR-Green I-based realtime RT-PCR assay for the detection and quantification of Chikungunya virus (CHIKV) in human, monkey and mosquito samples by targeting the E1 structural gene. A preliminary evaluation of this assay has been successfully completed using 71 samples, consisting of a panel of negative control sera, sera from healthy individuals, sera from patients with acute disease from which CHIKV had been isolated, as well as monkey sera and adult mosquito samples obtained during the chikungunya fever outbreak in Malaysia in 2008. The assay was found to be 100-fold more sensitive than the conventional RT-PCR with a detection limit of 4.12x10(0) RNA copies/μl. The specificity of the assay was tested against other related viruses such as Dengue (serotypes 1-4), Japanese encephalitis, Herpes Simplex, Parainfluenza, Sindbis, Ross River, Yellow fever and West Nile viruses. The sensitivity, specificity and efficiency of this assay were 100%, 100% and 96.8% respectively. This study on early diagnostics is of importance to all endemic countries, especially Malaysia, which has been facing increasingly frequent and bigger outbreaks due to this virus since 1999.

Matched MeSH terms: Haplorhini/virology*