Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya

BMC Infectious Diseases

Research Article

Barry W. Alto¹  Ozlem Yaren²  Kevin M. Bradley²  Zunyi Yang²  Steven A. Benner³  Priyanka V. Gangodkar⁴  Nikhil Phadke⁴  Kunal N. Patil⁴  Shatakshi R. Ranade⁴ 

[1] Florida Medical Entomology Laboratory, University of Florida, Vero Beach, FL, USA;Foundation for Applied Molecular Evolution (FfAME), Gainesville, FL, USA;Foundation for Applied Molecular Evolution (FfAME), Gainesville, FL, USA;Firebird Biomolecular Sciences LLC, Alachua, FL, USA;GenePath Dx (Causeway Healthcare), Pune, Maharashtra, India;

关键词: Point-of-care diagnostics;    Multiplexed isothermal amplification;    Zika detection;    Fluorescence read-out;    Sample preparation;    Mosquito surveillance;    Virus detection;   

DOI  :  10.1186/s12879-017-2382-0

 received in 2016-12-30, accepted in 2017-04-04,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundZika, 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.MethodsProcedures 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.ResultsThe 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.ConclusionsA 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.

【 授权许可】

CC BY   
© The Author(s). 2017