Researchers have developed a low-cost, rapid paper-based diagnostic system for strain-specific detection of the Zika virus, with the goal that it could soon be used in the field to screen blood, urine, or saliva samples.

They demonstrated a proof-of-concept color-changing diagnostic that could screen for Ebola by embedding in paper a novel kind of synthetic biomolecular sensor designed to screen for specific RNA sequences. These RNA sequences can mark not only the genetic signatures of Ebola but also other RNA viruses including Zika, SARS, measles, influenza, hepatitis C, and West Nile fever. The team believed that one day, the method could be applied in the field to identify viruses with blood, urine or saliva samples.

Researchers developed a simple modular workflow comprising three steps: amplification, Zika detection, and CRISPR-Cas9-aided strain identification. CRISPR-Cas9, a gene editing mechanism derived from the immune systems of bacteria, can be used to search entire sequences to find exclusive genetic markers. Leveraging CRISPR-Cas9's talent for sequence recognition, the third part of the team's system uses a CRISPR-Cas9-aided paper-based diagnostic to discriminate between strains whose genetic profiles differ by as little as one nucleotide.

Once a sample's RNA has been amplified using a mixture of enzymes and "primers", DNA sequences that trigger replication, a drop is administered to paper discs that are freeze-dried containing a mixture of cellular components and biological proteins. The droplet of amplified RNA activates the freeze-dried components so that the discs will change color to indicate a positive result for Zika virus. While the result can be read with the naked eye similar to a home pregnancy test, a specially designed electronic reader can also be used to get faster results and could, one day, quantify the amount of viral load in a sample.

If Zika is detected, the third step involves mixing a sample with a freeze-dried CRISPR-Cas9 cocktail and then using that mixture to wet another set of color-changing paper discs. Depending on the type of Zika strain contained in the sample, these discs undergo another set of visible color changes. Although synthetic biologists and genetic engineers usually put CRISPR-Cas9 to work inside living cells, Collins' team discovered that it functions just as well - and even better in some cases -- when freeze dried.

"We have tested our diagnostic systems against closely-related strains of the Dengue virus and found that within the first two steps, our system can readily distinguish Zika from Dengue," said co-first author on the study.

All components of the diagnostic system can be freeze-dried for storage and transport while retaining their efficacy. The ability to pinpoint a strain-specific diagnosis in the field could prove valuable to national and global health organizations for tracking the spread of a viral outbreak in real time and for preparing containment strategies and treatment plans.

http://wyss.harvard.edu/viewpressrelease/256