Simultaneous detection of three arboviruses using a triplex RT-PCR enzyme hybridization assay

Dong, Dan Hong; Fu, Shihong; Wang, Lihua; Li, Zhi; Li, Taiyuan; Liang, Guodong

doi:10.1007/s12250-012-3246-9

Cited by 10 publications

(11 citation statements)

References 18 publications

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“…; Dong et al . ), standard PCR‐based approaches require arrays of multiple assays to screen for broad diversities of viruses (Lwande et al . ; Ochieng et al .…”

Section: Discussionmentioning

confidence: 99%

“…In our laboratory, PCR-HRM costs less than $1 per sample, considering all consumables employed, after RNA extraction and reverse transcription. Although some methods may screen a few viruses in single-tube reactions at likely comparable costs (Chao et al 2007;Naze et al 2009;Dong et al 2013), standard PCR-based approaches require arrays of multiple assays to screen for broad diversities of viruses (Lwande et al 2013;Ochieng et al 2013;Liu et al 2016), and rely on sequencing of all samples (Lambert & Lanciotti 2009;Lwande et al 2013;Ochieng et al 2013), or require laborious PCR clean-up and mass spectrometric analysis (Briese et al 2005;Eshoo et al 2007). In contrast, multiplex PCR-HRM pathogen detection is less laborious, but has previously only been done on bacterial pathogens (Yang et al 2009;Tong & Giffard 2012;Xue et al 2012) and among certain flaviviruses (dengue serotypes 1-4, West Nile and chikungunya viruses) (Naze et al 2009).…”

Section: Discussionmentioning

confidence: 99%

“…) and triplex RT–PCR enzyme hybridization assay (Dong et al . ), have been used to screen for sets of specific arboviruses. Specific virus sequences can be differentiated among genus‐specific PCR amplicons by electrospray ionization mass spectrometry (Eshoo et al .…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, RT-PCR-based arbovirus identification is most commonly performed on sample cultures that generate cytopathic effects (CPE), as these provide sufficient template to screen with multiple tests (Lwande et al 2013;Ochieng et al 2013;Rosenberg et al 2013). Nonetheless, high-throughput multiplex approaches, such as MassTag PCR (Briese et al 2005), TaqMan Arrays (Chao et al 2007;Liu et al 2016) and triplex RT-PCR enzyme hybridization assay (Dong et al 2013), have been used to screen for sets of specific arboviruses. Specific virus sequences can be differentiated among genusspecific PCR amplicons by electrospray ionization mass spectrometry (Eshoo et al 2007) and by their high-resolution melting (HRM) profiles (Naze et al 2009;Omondi et al 2015).…”

Section: Introductionmentioning

confidence: 99%

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Arbovirus and insect‐specific virus discovery in Kenya by novel six genera multiplex high‐resolution melting analysis

Villinger

Mbaya

Ouso

et al. 2016

Molecular Ecology Resources

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A broad diversity of arthropod-borne viruses (arboviruses) of global health concern are endemic to East Africa, yet most surveillance efforts are limited to just a few key viral pathogens. Additionally, estimates of arbovirus diversity in the tropics are likely to be underestimated as their discovery has lagged significantly over past decades due to limitations in fast and sensitive arbovirus identification methods. Here, we developed a nearly pan-arbovirus detection assay that uses high-resolution melting (HRM) analysis of RT-PCR products from highly multiplexed assays to differentiate broad diversities of arboviruses. We differentiated 15 viral culture controls and seven additional synthetic viral DNA sequence controls, within Flavivirus, Alphavirus, Nairovirus, Phlebovirus, Orthobunyavirus and Thogotovirus genera. Among Bunyamwera, sindbis, dengue and Thogoto virus serial dilutions, detection by multiplex RT-PCR-HRM was comparable to the gold standard Vero cell plaque assays. We applied our low-cost method for enhanced broad-range pathogen surveillance from mosquito samples collected in Kenya and identified diverse insect-specific viruses, including a new clade in anopheline mosquitoes, and Wesselsbron virus, an arbovirus that can cause viral haemorrhagic fever in humans and has not previously been isolated in Kenya, in Culex spp. and Anopheles coustani mosquitoes. Our findings demonstrate how multiplex RT-PCR-HRM can identify novel viral diversities and potential disease threats that may not be included in pathogen detection panels of routine surveillance efforts. This approach can be adapted to other pathogens to enhance disease surveillance and pathogen discovery efforts, as well as the study of pathogen diversity and viral evolutionary ecology.

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“…; Dong et al . ), standard PCR‐based approaches require arrays of multiple assays to screen for broad diversities of viruses (Lwande et al . ; Ochieng et al .…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Arbovirus and insect‐specific virus discovery in Kenya by novel six genera multiplex high‐resolution melting analysis

Villinger

Mbaya

Ouso

et al. 2016

Molecular Ecology Resources

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“…A few reverse transcription polymerase chain reactions (RT-PCRs) have been developed for detection of GETV RNA by using different pairs of primers for differentiating among virus species. A *Corresponding author Tel: +82-54-912-0785, Fax: +82-54-912-0812 E-mail: yangdk@korea.kr triplex RT-PCR enzyme hybridization assay has been developed for simultaneous detection of GETV, Japanese encephalitis virus (JEV), and Tahyna virus [3]. In this RT-PCR assay, only clinical samples suspected of JEV infection were tested experimentally.…”

Section: Introductionmentioning

confidence: 99%

Establishment of reverse transcription polymerase chain reaction for detection of Getah virus infection in livestock

Lee¹,

Yang²,

Kim³

et al. 2017

Korean Journal of Veterinary Research

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Getah virus (GETV) infection causes sporadic outbreaks of mild febrile illness in horses and reproductive failure in pigs. In this study, we established a reverse transcription polymerase chain reaction (RT-PCR) method to detect GETV from suspected virus-infected samples. The reaction conditions were optimized and validated by using RNA extracted from GETV propagated in cell culture. A GETV-specific GED4 primer set was designed and used to amplify a 177 bp DNA fragment from a highly conserved region of the E1 glycoprotein gene in the GETV genome. RT-PCR performed with this primer set revealed high sensitivity and specificity. In the sensitivity test, the GED4 primer set detected GETV RNA at the level of 10 2.0 TCID 50 /mL. In the specificity test, the GED4 primer set amplified only a single band of PCR product on the GETV RNA template, without non-specific amplification, and exhibited no crossreactivity with other viral RNAs. These results suggest that this newly established RT-PCR method is useful for accurate identification of GETV infection in animals.

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Investigation of the genotype III to genotype I shift in Japanese encephalitis virus and the impact on human cases

et al. 2015

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Japanese encephalitis is a mosquito borne disease and is the leading cause of viral encephalitis in the Asia-Pacific area. The causative agent, Japanese encephalitis virus (JEV) can be phylogenetically classified into five genotypes based on nucleotide sequence. In recent years, genotype I (GI) has displaced genotype III (GIII) as the dominant lineage, but the mechanisms behind this displacement event requires elucidation. In an earlier study, we compared host variation over time between the two genotypes and observed that GI appears to have evolved to achieve more efficient infection in hosts in the replication cycle, with the tradeoff of reduced infectivity in secondary hosts such as humans. To further investigate this phenomenon, we collected JEV surveillance data on human cases and, together with sequence data, and generated genotype/case profiles from seven Asia-Pacific countries and regions to characterize the GI/GIII displacement event. We found that, when comprehensive and consistent vaccination and surveillance data was available, and the GIII to GI shift occurred within a well-defined time period, there was a statistically significant drop in JEV human cases. Our findings provide further support for the argument that GI is less effective in infecting humans, who represent a dead end host. However, experimental investigation is necessary to confirm this hypothesis. The study highlights the value of alternative approaches to investigation of epidemics, as well as the importance of effective data collection for disease surveillance and control.

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Simultaneous detection of three arboviruses using a triplex RT-PCR enzyme hybridization assay

Cited by 10 publications

References 18 publications

Arbovirus and insect‐specific virus discovery in Kenya by novel six genera multiplex high‐resolution melting analysis

Arbovirus and insect‐specific virus discovery in Kenya by novel six genera multiplex high‐resolution melting analysis

Establishment of reverse transcription polymerase chain reaction for detection of Getah virus infection in livestock

Investigation of the genotype III to genotype I shift in Japanese encephalitis virus and the impact on human cases

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