University of Pennsylvania engineers have developed a rapid, low-cost genetic test for the Zika virus.

The $2 testing device, about the size of a soda can, does not require electricity or technical expertise to use. A patient would simply provide a saliva sample, and color-changing dye turns blue if the genetic assay detects the presence of the virus.

Cutaway of the Zika test. Image credit: UPenn.Rapid, accurate diagnosis is especially important for pregnant women who may be infected. However, the only currently approved tests for the virus require highly sensitive laboratory equipment. Diagnostic tools that can be used in the field, while the patient waits, are potentially a critical tool for fighting the Zika epidemic.

Assays that detect genetic material from the Zika virus itself are considered the gold standard in diagnostics. Alternatives, such as tests that look for antibodies that the body creates in response to the virus, are insufficient as they may produce false negatives from people who are infected but haven't yet produced enough antibodies—or false positives from people who have antibodies for a different disease that is similar enough to trigger the test.

Tests that look for RNA sequences from the virus itself, known as reverse transcriptase polymerase chain reaction (RT-PCR) avoid both problems. However, RT-PCR requires delicate laboratory work. Viral gene sequences in a patient's sample must be amplified, or repeatedly copied, to levels where they can be detected, a process that normally involves multiple precise temperature changes.

With the amplification step as the main hurdle to a portable genetic test, Penn researchers led by Assistant Professor Changchun Liu and Professor Haim Bau, of the Department of Mechanical Engineering and Applied Mechanics, investigated the possibility of using an alternative technique known as reverse transcription loop-mediated isothermal amplification (RT-LAMP), which requires only that the sample be kept at a specific temperature, not cycled through multiple precise temperature changes as in RT-PCR.

As a trade-off for this simplified amplification process, RT-LAMP requires even more specialized "primers," short gene sequences that are designed to match the regions of the virus' DNA targeted by the test.

"Although Zika primers for RT-PCR have been published in the literature, RT-LAMP primers have not," says Bau. "So, using data mining, we identified highly conserved regions of the Zika virus genome that are divergent from other known pathogens. We then designed appropriate primers to recognize this sequence."

In parallel, the researchers engineered a low-cost, point-of-care system that consists of a diagnostic cassette and a processor. The cassette isolates, concentrates and purifies nucleic acids and carries out enzymatic amplification.

The researchers then worked on how to keep the sample at the necessary temperature without using electricity. Their solution involved a thermos bottle, a self-contained heating element that uses a chemical reaction from portable military rations and a wax-like material that absorbs excess heat by melting. A 3D-printed lid fits on top of the thermos and holds all of the test's components in place.

Once a patient's saliva sample is introduced into the cartridge, the test takes about 40 minutes to run.

Future work will demonstrate the test's selectivity and trial a version that can quantify the viral load by means of a fluorescent dye and an integrated smartphone camera.