A pocket-sized PCR test delivers results in 15 minutes

A pocket-sized PCR test delivers results in 15 minutes

Pandemics have affected humanity since prehistorical times, and their frequency is expected to increase due to an expanding population, global warming, and closer human-animal interactions. This presents a challenging scenario in disease prevention, detection, and response. 

Diagnostic tests, like the commonly used polymerase chain reaction (PCR), are essential public health tools for managing and controlling a pandemic as they provide a means for early detection, raising public awareness, monitoring variants, and helping to determine resource allocation.

“The most critical lesson from the COVID-19 pandemic is the importance of effective preventive public health via a rapid and reliable molecular diagnostic platform at home during outbreaks,” said Luke Lee, professor of medicine at Harvard Medical School. 

PCR is one of the most powerful diagnostic tests, recognizing specific sequences of DNA, and is the gold standard method for COVID-19 diagnosis due to its high sensitivity and flexibility.

But to give a speedy and secure response, PCR still needs to improve on this front. The reality is that while a conventional PCR test might only take 45 minutes to run, bottlenecks in our healthcare systems result in a significant amount of time from sampling to patients receiving their test results.

This is because PCR needs trained personnel, and people have to travel to healthcare centers for testing, which could cause further spread of the disease and additional strain on healthcare systems during a pandemic. This is what Lee was looking to change.

In a recent study, Lee and a group of scientists from Harvard Medical School, Sungkyunkwan University, and the Samsung Medical Center in South Korea sought to put the solution in the palm of our hands with a portable, easy-to-use PCR test on-a-chip.

The hand-held device was designed for use by the general public, providing diagnoses in only 15 minutes without the need for testing appointments and long wait times for results. They named it MEDIC-PCR and hope it, in addition to other meaningful public health policies, could contribute to preventing the spread of the infection by allowing people to safely test at home. 

How does PCR work?

DNA is made up of four building blocks: nucleotides called adenine (A), thymine (T), cytosine (C), and guanine (G), arranged in two strands that form a helix, held together through reversible molecular forces. But the pairing between these strands is not random: A always matches with T, while C pairs with G. This internal complementarity in DNA allows scientists to determine the order in one strand from its partner´s sequence. 

The PCR test identifies specific DNA sequences and amplifies their quantity using a polymerase enzyme, which builds new DNA molecules using each strand as a template. To separate the DNA strands to allow the enzyme access, scientists use heat followed by a cooling step that sets the optimal working temperature for the polymerase. This heating-cooling cycle is repeated between 30 and 40 times, and DNA amplification can be monitored in real time by adding a fluorescent dye that binds to DNA.

Since DNA duplicates after each cycle, the PCR can expand a single DNA molecule into more than a billion copies, allowing it to detect pathogens early in the infection when their quantity in swabs or samples is low. Moreover, as PCR is specific — it can identify DNA sequences within a mix of other nucleic acids present in the sample and discriminate between very similar DNA sequences, which means it can also identify different strains of the same virus.

Compacting the PCR test on a chip

To carry out PCR tests in a lab, scientists need specialized equipment, such as security cabins that prevent scientists from infecting themselves and a device called a thermocycler. This expertise and required lab equipment creates a barrier, especially in times of high demand like a pandemic.

Gyoujin Cho, professor of biophysics at Sungkyunkwan University and one of the lead authors of the study, told Advanced Science News that the project’s first and most challenging task was to compact the bulky components of the thermocycler onto a hand-held chip.

To do this, they used a 14 x 14 mm carbon black thin film that generates heat using an LED light as its energy source. “The incident photons from LED light at the carbon black [film], consisting of multi-layered graphene and amorphous carbon, interact with the free electrons in the carbon film and generate heat,” added the study’s co-author, Kiran Shrestha. This technology achieves stable heating and cooling cycles by simply turning the LED light on and off.

The team printed a carbon film onto transparent plastic, which serves as the chip’s support, and added the LED light source. Above the carbon film, they imprinted miniaturized slots with 1 μL capacities to carry out the PCR reactions and a small microscope to detect fluorescence and monitor the reaction.

“The low-cost carbon black film with [sampling wells are] manufactured through a roll-to-roll printing method,” explained Cho. This technique prints flexible structures, like microchips, with high quality and speed, allowing high production rates to respond rapidly in case of another pandemic.

Optimizing PCR on-a-chip

One of the most time-consuming steps of PCR testing is sample preparation. Samples from the nose and throat must be pretreated to break down virus particles and infected cells and release the viral genetic material. Scientists then purify the solution to avoid having other molecules in the mix that might interfere with the PCR reaction.

To simplify sample processing, the team eliminated the purification step, loading the pre-treated samples directly into the mini slots on the chip.

Some viruses, like the one responsible for COVID-19, use RNA instead of DNA to carry their genetic information. A step is, therefore, required to convert RNA into DNA to detect them with a PCR test. This is carried out by another enzyme called reverse transcriptase, which the scientists included in the reagents along with the polymerase enzyme.

Looking to reduce the PCR time, the scientists studied the interaction between the LED light and the carbon film. Testing different carbon film thicknesses and LED light wavelengths, they found that a film of 14 µm and LED light of 940 nm was the combination with the highest light-to-heat conversion efficiency. 

Under these conditions, they achieved a heating rate of 22°C per second and a cooling rate of -2.5°C per second. While the cooling rate is similar to a PCR made with a classical thermocycler at the lab, the heating rate is seven times higher.

“In general, to test COVID-19 using the conventional PCR method at a certified place usually takes about six hours, from sample collection and transport to the PCR lab, to the result,” said Seongryeong Kim, graduate student and co-author of the study. “However, due to the reduction of sample preparation and PCR on-a-chip, our MEDIC-PCR takes only 15 minutes.”

Validating MEDIC-PCR

To test the performance of MEDIC-PCR, the team first assessed if it could detect purified RNA from SARS-CoV-2. The MEDIC-PCR responded positively to growing RNA concentrations and performed similarly to a conventional PCR test. It also showed high sensitivity as it detected the purified viral RNA in all 50 samples containing a fixed RNA concentration. 

Finally, the scientists challenged the device with clinical samples to check how well the MEDIC-PCR identified positive and negative cases in the context of infection, where the virus is complete and in its natural environment.

A reliable PCR test would detect infected and non-infected individuals with minimal mistakes. The MEDIC-PCR showed an impressive diagnostic accuracy of 97%, correctly identifying the infection in 84 out of 89 infected patients and only identifying one person as positive in 103 individuals who were not infected.

“Although we used only a single target gene to test for COVID in this paper, four simultaneous PCR [reactions] can be carried out, which would allow to detect four different target genes,” wrote the researchers in their study.

This means that the MEDIC-PCR device could have broader use than the one tested in this study, allowing the detection of different genes of one pathogen, or even different pathogens or biomarkers simultaneously, making it suitable for diagnosing other diseases.

Besides preventing disease transmission by keeping people informed and allowing them to stay at home when infected, this portable PCR device enables people to participate more actively in disease surveillance by remotely reporting the results to healthcare agencies.

According to the authors, the estimated price of the MEDIC-PCR is $1.26 USD per test, taking into account only the reagents and chip materials. The team hopes the easy manufacturing and low cost will make this an accessible and powerful public health tool.

Reference: Kiran Shrestha et al., Mobile efficient diagnostics of infectious diseases via on-chip RT-qPCR: MEDIC-PCR, Advanced Science (2023). DOI: 10.1002/advs.202302072

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