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AI Biosciences Thermos Thermal Cycler Adapted for Multiplexing, RT-PCR

Setup of the TTC.

NEW YORK (GenomeWeb) – A thermal cycler built for less than $200 using thermoses and a servo arm has been shown capable of detecting infectious disease agents with cycle times of 15 to 30 seconds.

AI Biosciences previously developed the low-cost, rapid, thermos thermal cycler (TTC) and published proof-of-principle cycle times, as reported by GenomeWeb.

Now, in a study published in PLOS One the firm has increased the number of samples that can be processed simultaneously to eight, demonstrated multiplexing capability of the system using a commercial assay, and enabled RT-PCR via the TTC method while validating it on various infectious disease targets using standard protocols and reagents.

Season Wong, co-founder and president of AI Biosciences, told GenomeWeb that the new configuration uses an additional thermos for the reverse-transcriptase step.

"We have previously used two thermoses to perform two-step PCR for DNA," he explained, adding, "Conceptually, only another Thermos is needed [for RT-PCR], but we felt the need to prove that, since many pathogens are RNA-based."

The firm also aims to develop the method further in the future to add real-time detection capability, he said.

Briefly, the thermal cycler works by using a battery-operated Arduino-based microcontroller mounted onto a soup can, with a wire and a stick fashioned into a dunking arm. The temperature in the denaturation bath is maintained above 90 degrees by pouring boiling water into the thermos containing 11 milliliters of oil. A mathematical equation describes the proportion of room temperature water that must be added to boiling water to achieve any desired annealing and extension bath temperature in additional thermoses.

For the most recent study, AI Biosciences researchers collaborated with Charlotte Gaydos and Justin Hardick at Johns Hopkins University, who contributed clinical urine samples of patients known to be infected with chlamydia and suggested comparator assays. They also offered insights on how rapid molecular tests for STIs should ideally operate so that they meet the needs of users and patients, Wong noted.

Gaydos recently validated a Trichomonas vaginalis test from Quidel on the AmpliVue platform and has collaborated with Atlas Genetics on a point-of-care chlamydia assay that was CE-marked earlier this month. Hardick, meanwhile, published a clinical validation of the Abbott Iridica system for influenza diagnosis last year, as covered by GenomeWeb.

For the clinical chlamydia samples, the TTC was able to complete detection in less than 12 minutes, compared to 45 minutes for commercial PCR reactions.

AI Biosciences previously received Phase I Small Business Innovation Research grants from the National Institutes of Health to develop rapid point-of-care tests for STDs and dengue, Wong said, and in the PLOS One study the platform was also validated on dengue virus I.

The researchers also chose additional targets to further demonstrate the value of the TTC amplification of RNA targets by RT-PCR, Wong said. These targets included extracted HIV-1 RNA and recombinant Ebola virus RNA. For the latter, fewer than 10 template copies of RNA were detectable based on the Cq values, supporting the idea that "the efficiency of the TTC is very close to that of a commercial unit."

The price to build the new configuration is higher than the previous one due primarily to the cost of adding a third thermos, Wong said. In some PCR runs, the firm also tried using larger thermoses of 24 oz versus 16 oz, since they maintain temperature slightly better due to larger thermal mass, and cost only a few more dollars to purchase. Regardless, the current $200 price tag remains significantly less than a typical thermal cycler.

And, since the temperature of the water is maintained using a layer of oil, the device essentially runs using only the energy it takes to boil water — an estimated 0.44 kilowatt hours of electricity for 10 consecutive runs at an approximate cost of about $.05 in the US or $.08 in Nigeria, according to the study.

AI Biosciences is currently receiving Phase I SBIR funding to develop a rapid test for norovirus, Wong said. The company is considering the possibility of additional funding from non-profit organizations and is also exploring developing the TTC concept into a STEM education lesson plan. "Students can learn to apply their math, physics, biology, and programming knowledge to construction of the TTC; it would be a fun project ... [and] we can also get user feedback from students on their experience using the TTC," Wong said.

The firm also has a few partners in academia and is looking to work with industry partners in the near future.

In terms of intellectual property, it has filed a provisional patent on the TTC and has other IP plans that it is not ready to disclose at this time. 

"Our company is focused on developing point-of-care molecular diagnostics for low-resource settings, so the work we do carries the risk of being too simple and could be copied by others if the idea is revealed," said Wong.