A team of international, multi-institutional researchers led by Wyss Institute for Biologically Inspired Engineering at Harvard University synthetic biologist James Collins has developed a rapid paper-based diagnostic system for strain-specific detection of the Zika virus.
Claimed to be low-cost, the diagnostic system aims can apply the test in the field to test blood, urine, or saliva samples.
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In the wake of a rapid spread of the Ebola virus, Collins and his team, along with collaborators from Massachusetts Institute of Technology (MIT), the Broad Institute of Harvard and MIT, Harvard Medical School (HMS), University of Toronto, Arizona State University (ASU), University of Wisconsin-Madison (UW-Madison), Boston University (BU), Cornell University, and Addgene, in 2014, developed the diagnostic test for embedding synthetic gene networks.
It was a proof-of-concept, colour-changing diagnostic that could screen for Ebola by embedding in paper a new kind of synthetic biomolecular sensor designed to screen for specific RNA sequences.
The RNA sequences can reflect the genetic signatures of Ebola along with other RNA viruses including Zika, SARS, measles, influenza, hepatitis C, and West Nile fever.
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However, the earlier paper-based technology could not be used on samples like blood, urine, and saliva owing to the lesser concentration of virus in the samples.
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By GlobalDataThe product overcame this initial challenge after it had been administered on blood samples of a monkey infected by Zika virus, as well as virus recovered from cells infected in the laboratory.
The diagnostic system involves an easy modular workflow comprising of three steps: amplification, Zika detection, and CRISPR-Cas9-aided strain identification.
CRISPR-Cas9 is a gene editing mechanism exacted from the immune systems of bacteria that can be used to search entire sequences to find exclusive genetic markers.
It has been used by the team into the system 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 change colour to indicate the presence of Zika virus.
The result can be interpreted with the naked eye, as well as a specially designed electronic reader that can be used to get faster results and can count the amount of viral load in a sample in a day.
Upon the detection of Zika, the third step involves mixing a sample with a freeze-dried CRISPR-Cas9 cocktail and then using that mixture to wet another set of colour-changing paper discs.
Depending on the type of Zika strain contained in the sample, these discs undergo another set of visible colour changes. Although synthetic biologists and genetic engineers usually put CRISPR-Cas9 to work inside living cells, Collins’ team discovered that it functions even when freeze dried.
Wyss Institute and BU former postdoctoral fellow ASU’s biodesign institute and school of molecular sciences centre for molecular design and biomimetics assistant professor and study co-first author Alexander Green said: "The addition of the third CRISPR-based step deploying Cas9 on a paper-based platform for the first time only enhances the accuracy of detection.
"As we prepare this technology for translation, we plan to validate our system against dozens or even hundreds of clinical samples."
The diagnostic system can be freeze-dried for storage and transport while retaining their efficacy.
National and global health organisations will benefit from the ability to pinpoint a strain-specific diagnosis in the field, allowing them to track the spread of a viral outbreak in real time and prepare containment strategies and treatment plans.
The diagnostic system developed by Collins’ team can be customised to identify a range of pathogens.
The method can be used to quickly respond and develop new diagnostics in the face of emerging outbreaks.
Image: Paper-based diagnostic test to detect Zika virus. Photo: courtesy of Wyss Institute at Harvard University.
