Rapid Molecular Assays Detects Yellow Fever Virus

Rapid and reliable diagnostic methods that are simple to execute could have a major impact on early detection of outbreaks and implementation of appropriate response strategies such as vaccination and/or vector control.

Scientists at the Robert Koch Institute (Berlin, Germany) working with an international team of collaborators, adopted an isothermal approach and developed a recombinase polymerase amplification (RPA) assay which can be performed with a small portable instrument and easy-to-use lyophilized reagents. The assay was developed in three different formats, real-time with or without microfluidic semi-automated system and lateral-flow assay to evaluate their application for different purposes.

Viral ribonucleic acid (RNA) was isolated from of cell culture supernatants or aliquots of mosquito pools, using the QIAamp Viral Mini Kit (QIAGEN Lake Constance GmbH; Stockach, Germany). The assay to detect and quantify genomic RNA of YFV was performed in a one-step format on the ABI 7500 instrument (Life Technologies; Carlsbad, CA, USA) using the Qiagen’s QuantiTect Virus Kit. Lateral-flow strip real time (RT)-RPA assay was performed using the TwistAmp nfo RT kit from TwistDx (Cambridge, UK). Centrifugal microfluidic cartridges, termed GeneSlice (HSG-IMIT, Lab on a Chip Design- and Foundry Service; Freiburg, Germany), were used to demonstrate process automation of real-time RT-RPA in a small and portable processing device, the “SONDE” player, that may be used in the field with minimum manual interaction.


The authors found that the RT-RPA assay for YFV detection can be performed without complex equipment in a basic laboratory setting, a rural health care center or an outbreak field investigation. Real-time RT-RPA results demonstrated an optimal specificity. The authors concluded that the real-time RT-RPA assay, using the transportable Tube Scanner device combined with the RNA extraction method based on magnetic beads, and the use of lyophilized reagents which can be stored at ambient temperature allowed the application of the RPA assay under field conditions in Senegal with performance similar to that of cutting-edge laboratory settings. The study was published on March 6, 2014, in the journal Public Library of Science Neglected Tropical Diseases.

Related Links:
Robert Koch Institute 
QIAGEN
Life Technologies