Nucleic-acid detection is crucial for basic research as well as for applications in medicine such as diagnostics. In resource-limited settings, however, most DNA-detection diagnostic schemes are inapplicable since they rely on expensive machinery, electricity, and trained personnel. Here, we present an isothermal DNA detection scheme for the diagnosis of pathogenic DNA in resource-limited settings. DNA was extracted from urine and blood samples using two different instrument-free methods, and amplified using Recombinase Polymerase Amplification with a sensitivity of <10 copies of DNA within 15 minutes. Target DNA was bound by dCas9/sgRNA that was labelled with a DNA oligomer to subsequently induce Rolling Circle Amplification. This second amplification step produced many copies of a G-quadruplex DNA structure that facilitates a colorimetric readout that is visible to the naked eye. This isothermal DNA-detection scheme can be performed at temperatures between 20-45 °C. As an example of the applicability of the approach, we isothermally (23 °C) detected DNA from a parasite causing visceral leishmaniasis that was spiked into buffer and resulted in a sensitivity of at least 1 zeptomole. For proof of principle, DNA spiked into blood was coupled to the CRISPR-dCas9-based detection scheme yielding a colorimetric readout visible to the naked eye. Given the versatility of the guide-RNA programmability of targets, we envision that this DNA detection scheme can be adapted to detect any DNA with minimal means, which facilitates applications such as point-of-care diagnostics in resource-limited settings.

Inadequate and nonintegrated diagnostics are the Achilles' heel of global efforts to monitor, control, and eradicate neglected tropical diseases (NTDs). While treatment is often available, NTDs are endemic among marginalized populations, due to the unavailability or inadequacy of diagnostic tests that cause empirical misdiagnoses. The need of the hour is early diagnosis at the point-of-care (PoC) of NTD patients. Here, we review the status quo of PoC diagnostic tests and practices for all of the 24 NTDs identified in the World Health Organization's (WHO) 2021-2030 roadmap, based on their different diagnostic requirements. We discuss the capabilities and shortcomings of current diagnostic tests, identify diagnostic needs, and formulate prerequisites of relevant PoC tests. Next to technical requirements, we stress the importance of availability and awareness programs for establishing PoC tests that fit endemic resource-limited settings. Better understanding of NTD diagnostics will pave the path for setting realistic goals for healthcare in areas with minimal resources, thereby alleviating the global healthcare burden.

The rapid growth of point-of-care (POC) diagnostic tests necessitates a clear vision of when, where, and why a new POC diagnostic test needs to be developed and how it can be used in a way that matches a local health care context. Here, we present an innovative approach toward developing a concept target product profile (CTPP), which is a new mapping tool that helps researchers match a new diagnostic test to a specific local health care context early in the research and development process. As a case study, we focus on the diagnosis of visceral leishmaniasis (VL) in rural resource-limited regions of Kenya and Uganda. Our stepwise approach integrates elements of design thinking and uses a combination of literature reviews and field research for a context analysis of local health care systems and practices. We then use visual thinking in the form of Gigamaps and patient journeys to identify use case scenarios and to present our findings from the field research to key stakeholders. The use case scenarios describe the diagnostic scope of a new POC test based on the feasibility of the new test, the local need, and the contextual fit. For our case study of VL, we identify 2 valuable use case scenarios, namely test-of-cure and screening and confirmation, and we formulate a CTPP. We anticipate that a CTPP will enable researchers to match a new POC diagnostic test during the research and development process to the local health care context in which it will be used.

Solid-state nanopores have emerged as promising platforms for biosensing including diagnostics for disease detection. Here we show nanopore experiments that detect CRISPR-dCas9, a sequence-specific RNA-guided protein system that specifically binds to a target DNA sequence. While CRISPR-Cas9 is acclaimed for its gene editing potential, the CRISPR-dCas9 variant employed here does not cut DNA but instead remains tightly bound at a user-defined binding site, thus providing an excellent target for biosensing. In our nanopore experiments, we observe the CRISPR-dCas9 proteins as local spikes that appear on top of the ionic current blockade signal of DNA molecules that translocate through the nanopore. The proteins exhibit a pronounced blockade signal that allows for facile identification of the targeted sequence. Even at the high salt conditions (1 M LiCl) required for nanopore experiments, dCas9 proteins are found to remain stably bound. The binding position of the target sequence can be read from the spike position along the DNA signal. We anticipate applications of this nanopore-based CRISPR-dCas9 biosensing approach in DNA-typing based diagnostics such as quick disease-strain identification, antibiotic-resistance detection, and genome typing.