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New Zika Test Uses Isothermal RNA Amplification for Detection, CRISPR/Cas9 for Strain ID


NEW YORK (GenomeWeb) – A new point-of-care molecular test for Zika virus uses isothermal RNA amplification for detection and CRISPR/Cas9 to determine the particular strain.

Led by senior author Jim Collins of the Massachusetts Institute of Technology and a phalanx of co-first authors from the University of Toronto, Arizona State University, MIT, and Harvard University's Wyss Institute for Biologically Inspired Engineering, the multi-institutional team described the test in a paper published today in Cell.

"It's a freeze-dried, paper-based, synthetic biology platform" for detecting Zika virus, Collins told GenomeWeb. And as Zika sweeps through the Americas, the test could provide an important way to monitor the virus' spread. "It's cheap, rapid, and it can be modified quickly as strains arrive or mutate," Collins said.

Nucleic acid sequence-based amplification (NASBA) enables isothermal detection of Zika RNA in approximately two hours. The test also leverages technology called a toehold switch, a repressed messenger RNA that turns on translation of a gene when the switch encounters a specific RNA sequence. The scientists were able to engineer 24 different sensors targeting 24 regions of Zika RNA.

To add a strain identification step, the scientists designed a CRISPR/Cas9 system with guide RNAs targeting strain-specific regions of DNA to inhibit the toehold reaction.

Collins said the origins of the test lie in antibiotic-resistance detection, but the scientists leveraged the paper-based diagnostic for viral detection in the last throes of 2014's Ebola crisis. As reported by GenomeWeb, the group published the details of their Ebola test in Cell in October of that year.

"It was the tail end of the crisis, so we didn't move to get it in the field," Collins said, "but now we're at the front end of the Zika crisis."

Collins' team isn't alone in pivoting to Zika detection. The list of outfits pursuing Zika tests includes Quest Diagnostics, Roche, Ubiquitome, the US Centers for Disease Control and Prevention, Houston Methodist and Texas Children's hospitals, Siemens Healthcare Diagnostics, Bioneer, Luminex and Genarraytion, Rheonix, and Thermo Fisher Scientific.

The US Food and Drug Administration has already granted Emergency Use Authorization for the Quest and CDC tests, while Siemens is seeking that designation. The Bioneer test has received CE-IVD marking.

But the new test from Collins' team offers rapid results and ease of use that could bring it out of the lab and into the field.

The Zika test works in the same way as the Ebola test. After sample collection and RNA extraction, on-paper NASBA amplifies RNA through a process of reverse transcription, RNase H degradation, double-stranded DNA synthesis, and T7 RNA transcription. If Zika RNA is present in the sample, the toehold switches will quickly reveal it by turning the paper purple. From start to finish, the test takes only a shade over three hours to yield a result.

The scientists validated the test on human serum spiked with active Zika virus and three different Dengue virus serotypes, as well as with plasma samples from Rhesus macaques infected with Zika.

While the Ebola test used different toehold switch designs to differentiate between the Sudan and Zaire strains (the Ebola nucleoprotein differs by only three nucleotides between the two), the Zika test includes a new CRISPR-based method for strain ID.

Like the other reagents for the detection step, the CRISPR/Cas9 system is freeze dried onto the paper strips. While Collins can't be sure his team was the first to freeze dry CRISPR, "it was welcome news to us it would work," he said.

In its current form, the test requires an additional reaction for strain identification once Zika is detected from a sample. With CRISPR/Cas9 added to the mix the second time around, and with guide RNAs targeting the regions that differ between strains, the toehold switch reaction won't proceed if the Cas9-gRNA complex detects the target and successfully cleaves it. The toehold simply won't recognize its own RNA target and won't kick off reporter signal.

In the new paper, the scientists designed the CRISPR system to target the American strain; a negative result (no color change) would indicate identification of that strain. This works because the American and African Zika strains have diverged in a manner that has both created and destroyed CRISPR/Cas9 protospacer-adjacent motifs (PAMs) in either strain.

As shown in the Cell study, a SNP in a "CGG" in the American strain manifests as "CAG" in the African. The "CGG" in the American strain is a CRISPR/Cas9 recognition site ("NGG" being the canonical PAM for Cas9 from Streptococcus pyogenes), thus, a guide RNA targeting the 20 nucleotides upstream of the PAM can cleave DNA reverse-transcribed from American Zika RNA, but will likely fail with the African strain. "There are many strain-specific PAM sites that can be used for lineage discrimination," the authors wrote.

While the Cell paper presents only a proof-of-concept, Collins said he is looking for partners to help take the next step and begin scaling and manufacturing the test. "We've been talking with groups to get this deployed," he said, mentioning that non-governmental organizations might be an attractive partner, especially since the cost would enable large-scale monitoring of the virus' spread.

"NGOs like the World Health Organization can use this information to get ahead of an outbreak in order to contain it and save lives," he said.