Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya
Abstract Background Zika, dengue, and chikungunya are three mosquito-borne viruses having overlapping transmission vectors. They cause diseases having similar symptoms in human patients, but requiring different immediate management steps. Therefore, rapid (< one hour) discrimination of these thre...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2017-04-01
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| Series: | BMC Infectious Diseases |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12879-017-2382-0 |
| _version_ | 1819016561359323136 |
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| author | Ozlem Yaren Barry W. Alto Priyanka V. Gangodkar Shatakshi R. Ranade Kunal N. Patil Kevin M. Bradley Zunyi Yang Nikhil Phadke Steven A. Benner |
| author_facet | Ozlem Yaren Barry W. Alto Priyanka V. Gangodkar Shatakshi R. Ranade Kunal N. Patil Kevin M. Bradley Zunyi Yang Nikhil Phadke Steven A. Benner |
| author_sort | Ozlem Yaren |
| collection | DOAJ |
| description | Abstract Background Zika, dengue, and chikungunya are three mosquito-borne viruses having overlapping transmission vectors. They cause diseases having similar symptoms in human patients, but requiring different immediate management steps. Therefore, rapid (< one hour) discrimination of these three viruses in patient samples and trapped mosquitoes is needed. The need for speed precludes any assay that requires complex up-front sample preparation, such as extraction of nucleic acids from the sample. Also precluded in robust point-of-sampling assays is downstream release of the amplicon mixture, as this risks contamination of future samples that will give false positives. Methods Procedures are reported that directly test urine and plasma (for patient diagnostics) or crushed mosquito carcasses (for environmental surveillance). Carcasses are captured on paper samples carrying quaternary ammonium groups (Q-paper), which may be directly introduced into the assay. To avoid the time and instrumentation requirements of PCR, the procedure uses loop-mediated isothermal amplification (LAMP). Downstream detection is done in sealed tubes, with dTTP-dUTP mixtures in the LAMP with a thermolabile uracil DNA glycosylase (UDG); this offers a second mechanism to prevent forward contamination. Reverse transcription LAMP (RT-LAMP) reagents are distributed dry without requiring a continuous chain of refrigeration. Results The tests detect viral RNA in unprocessed urine and other biological samples, distinguishing Zika, chikungunya, and dengue in urine and in mosquitoes infected with live Zika and chikungunya viruses. The limits of detection (LODs) are ~0.71 pfu equivalent viral RNAs for Zika, ~1.22 pfu equivalent viral RNAs for dengue, and ~38 copies of chikungunya viral RNA. A handheld, battery-powered device with an orange filter was constructed to visualize the output. Preliminary data showed that this architecture, working with pre-prepared tubes holding lyophilized reagent/enzyme mixtures and shipped without a chain of refrigeration, also worked with human plasma samples to detect chikungunya and dengue in Pune, India. Conclusions A kit, complete with a visualization device, is now available for point-of-sampling detection of Zika, chikungunya, and dengue. The assay output is read in ca. 30 min by visualizing (human eye) three-color coded fluorescence signals. Assay in dried format allows it to be run in low-resource environments. |
| first_indexed | 2024-12-21T02:49:35Z |
| format | Article |
| id | doaj.art-dd0dfae12e7d4ababd79f7e0fe40225a |
| institution | Directory Open Access Journal |
| issn | 1471-2334 |
| language | English |
| last_indexed | 2024-12-21T02:49:35Z |
| publishDate | 2017-04-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Infectious Diseases |
| spelling | doaj.art-dd0dfae12e7d4ababd79f7e0fe40225a2022-12-21T19:18:29ZengBMCBMC Infectious Diseases1471-23342017-04-0117111310.1186/s12879-017-2382-0Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunyaOzlem Yaren0Barry W. Alto1Priyanka V. Gangodkar2Shatakshi R. Ranade3Kunal N. Patil4Kevin M. Bradley5Zunyi Yang6Nikhil Phadke7Steven A. Benner8Foundation for Applied Molecular Evolution (FfAME)Florida Medical Entomology Laboratory, University of FloridaGenePath Dx (Causeway Healthcare)GenePath Dx (Causeway Healthcare)GenePath Dx (Causeway Healthcare)Foundation for Applied Molecular Evolution (FfAME)Foundation for Applied Molecular Evolution (FfAME)GenePath Dx (Causeway Healthcare)Foundation for Applied Molecular Evolution (FfAME)Abstract Background Zika, dengue, and chikungunya are three mosquito-borne viruses having overlapping transmission vectors. They cause diseases having similar symptoms in human patients, but requiring different immediate management steps. Therefore, rapid (< one hour) discrimination of these three viruses in patient samples and trapped mosquitoes is needed. The need for speed precludes any assay that requires complex up-front sample preparation, such as extraction of nucleic acids from the sample. Also precluded in robust point-of-sampling assays is downstream release of the amplicon mixture, as this risks contamination of future samples that will give false positives. Methods Procedures are reported that directly test urine and plasma (for patient diagnostics) or crushed mosquito carcasses (for environmental surveillance). Carcasses are captured on paper samples carrying quaternary ammonium groups (Q-paper), which may be directly introduced into the assay. To avoid the time and instrumentation requirements of PCR, the procedure uses loop-mediated isothermal amplification (LAMP). Downstream detection is done in sealed tubes, with dTTP-dUTP mixtures in the LAMP with a thermolabile uracil DNA glycosylase (UDG); this offers a second mechanism to prevent forward contamination. Reverse transcription LAMP (RT-LAMP) reagents are distributed dry without requiring a continuous chain of refrigeration. Results The tests detect viral RNA in unprocessed urine and other biological samples, distinguishing Zika, chikungunya, and dengue in urine and in mosquitoes infected with live Zika and chikungunya viruses. The limits of detection (LODs) are ~0.71 pfu equivalent viral RNAs for Zika, ~1.22 pfu equivalent viral RNAs for dengue, and ~38 copies of chikungunya viral RNA. A handheld, battery-powered device with an orange filter was constructed to visualize the output. Preliminary data showed that this architecture, working with pre-prepared tubes holding lyophilized reagent/enzyme mixtures and shipped without a chain of refrigeration, also worked with human plasma samples to detect chikungunya and dengue in Pune, India. Conclusions A kit, complete with a visualization device, is now available for point-of-sampling detection of Zika, chikungunya, and dengue. The assay output is read in ca. 30 min by visualizing (human eye) three-color coded fluorescence signals. Assay in dried format allows it to be run in low-resource environments.http://link.springer.com/article/10.1186/s12879-017-2382-0Point-of-care diagnosticsMultiplexed isothermal amplificationZika detectionFluorescence read-outSample preparationMosquito surveillance |
| spellingShingle | Ozlem Yaren Barry W. Alto Priyanka V. Gangodkar Shatakshi R. Ranade Kunal N. Patil Kevin M. Bradley Zunyi Yang Nikhil Phadke Steven A. Benner Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya BMC Infectious Diseases Point-of-care diagnostics Multiplexed isothermal amplification Zika detection Fluorescence read-out Sample preparation Mosquito surveillance |
| title | Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya |
| title_full | Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya |
| title_fullStr | Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya |
| title_full_unstemmed | Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya |
| title_short | Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya |
| title_sort | point of sampling detection of zika virus within a multiplexed kit capable of detecting dengue and chikungunya |
| topic | Point-of-care diagnostics Multiplexed isothermal amplification Zika detection Fluorescence read-out Sample preparation Mosquito surveillance |
| url | http://link.springer.com/article/10.1186/s12879-017-2382-0 |
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