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Researchers Develop Low-Cost Pan-HPV MDx Using Enzyme-DNA Nanostructures


NEW YORK (360Dx) – Researchers at the National University of Singapore have developed a rapid pathogen detection platform that uses microfluidics containing integrated circuits of DNA nanostructures.

Called enzyme-assisted nanocomplexes for visual identification of nucleic acids, or enVision, the system has been shown to be capable of room temperature molecular typing of human papillomavirus from cervical samples, as well as discovering certain infections that can be undetectable by most standard methods.

The enVision platform was developed at the NUS Biomedical Institute for Global Health Research and Technology, also called BIGHEART, by Huilin Shao and her team. The core technology of the modular, "plug-and-play" platform involves DNA-enzyme nanostructures consisting of complementary DNA bound to polymerase and self-priming DNA nanostructures that serve as universal signaling elements.

Although it is only in prototype form, the device was shown in a recent proof-of-concept Nature Communications study to be more sensitive than qPCR-based methods. It could also detect target DNA at attomolar concentrations at a cost of around S$1 (US$1.38) per test.

"Molecular diagnostics can provide vitally important information to patients and physicians," Shao said in an email, and so she and her BIGHEART team have been focused on developing sensitive and inexpensive test chips that work robustly at room temperature in order "to democratize molecular diagnostics."

The group chose to tackle HPV – the leading cause of cervical cancer – for initial assay development.

Although the entire HPV genome can be inserted into the host genome during infection, partial genome integrations are possible and can also lead to cancer, Shao explained. Many commercial assays detect a locus of the HPV genome called L1, but detecting other regions, such as the L2 or E1 loci, could help to capture partial integrations, Shao said, "leading to better coverage for infection determination and management."

EnVision was designed specifically to decouple target recognition from signaling, to enable programmable detection and logic computations.

The DNA-enzyme nanostructures are complexes made of inactivating aptamers linked to Taq DNA polymerase. When complementary target DNA binds an aptamer, the polymerase is released. The freed polymerases then use biotinylated dNTPs circulating in the microfluidic cassette to elongate a nearby signaling nanostructure made from a self-priming hairpin molecule.

The biotinylated dNTPs in the signaling structure will also bind streptavidin-horseradish peroxidase (HRP) molecules in the reaction chamber, and in the presence of diaminobenzidine peroxidase the HRP produces a brown precipitate that can be seen with the naked eye or quantified with a smartphone.

The system is also modular. Detection reactions take place in an independent microfluidic assay cassette that is preloaded with nanostructures. In order to perform the assay, a test cassette is mounted to a separate cartridge module that is the same for all tests, containing membranes embedded with the universal signaling nanostructures.

Compared to qPCR, enVision showed better sensitivity and fewer false-positive results on a set of synthetic targets representing different subtypes of HPV. Shao highlighted that qPCR can be prone to false positives due to non-specific amplification and formation of primer dimers, which, in a clinical setting, can lead to misdiagnoses, wrong or delayed treatments, and patient anxiety and poor health outcomes.

There are more than 100 subtypes of HPV, but only approximately 15 are considered to be highly carcinogenic, with HPV 16 and 18 subtypes most commonly tested for. Although subtyping is not typically performed, it could potentially be useful for epidemiology as well as patient stratification and monitoring. The recognition element in the enVision test is easily adjustable, so it could detect any subtypes required or be designed to only produce a signal when multiple sequences or loci are present. 

The enVision test was also run on a set of 35 clinical endocervical samples in the study and compared to a gold standard test, the Roche Cobas qPCR-based HPV assay.

In this case, the authors set up enVision to detect HPV 16 and HPV 18 L1 loci in the patient genome, which are the targets of the Roche test. EnVision achieved about 93 percent sensitivity and 91 percent specificity for HPV 16, and for HPV 18 the assay showed 83 percent sensitivity and 100 percent specificity compared to the Cobas test. Notably, this level of sensitivity and specificity on clinical samples was seen without any pre-amplification, in an equipment-free assay performed at room temperature.

Shao and her group also designed a high-coverage, multi-loci test and evaluated it with the same set of clinical samples. They discovered L2 and E1 integration in a subset of L1-negative samples, which was confirmed by TaqMan fluorescence analysis, and concluded that a multi-loci enVision test could potentially improve detection coverage and identify previously undetectable infections.

The device currently requires nucleic acid extraction from biological samples, but RNA can be detected directly on the platform without requiring conversion to DNA, Shao said. "We thus foresee that the sample prep can be easily streamlined on the enVision platform," she said.

Shao and her team are now developing an integrated sample prep module for point-of-care applications.

They are also investigating using isothermal amplification to pre-amplify the nucleic acids and improve sensitivity. In the study, the team compared enVision to a loop-mediated isothermal amplification (LAMP) test and observed that LAMP is prone to primer-dimer formation and false-positive results. However, an isothermal technology called nucleic acid sequence-based amplification, or NASBA, may improve the detection sensitivity "without sacrificing the specificity conferred by the enVision recognition nanostructures," Shao said. The group is currently testing this and other technologies for functionality at room temperature and hopes to incorporate them into future iterations of enVision.

The group is also pursuing detection of other infectious diseases such as dengue virus and malaria. "Beyond infectious diseases, we also hope to expand the system to detect other diseases, such as genetic abnormalities, to enable personalized medicine," Shao said, adding, "We are working on improving the chip design to make it foolproof for widespread use near patients."

In the near-term, the BIGHEART group also expects to undertake larger clinical validation studies. Shao said the team is currently in discussion with several interested companies, but added that "spinning out on our own also presents an impactful opportunity."