RNA detection with high specificity and sensitivity using nested fluorogenic Mango NASBA

Abstract

There is a pressing need for nucleic acid-based assays that are capable of rapidly and reliably detecting pathogenic organisms. Many of the techniques available for the detection of pathogenic RNA possess one or more limiting factors that make the detection of low-copy RNA challenging. Although RT-PCR is the most commonly used method for detecting pathogenrelated RNA, it requires expensive thermocycling equipment and is comparatively slow. Isothermal methods promise procedural simplicity but have traditionally suffered from amplification artifacts that tend to preclude easy identification of target nucleic acids. Recently, the isothermal SHERLOCK system overcame this problem by using CRISPR to distinguish amplified target sequences from artifactual background signal. However, this system comes at the cost of introducing considerable enzymatic complexity and a corresponding increase in total assay time. Therefore, simpler and less expensive strategies are highly desirable. Here, we demonstrate that by nesting NASBA primers and modifying the NASBA inner primers to encode an RNA Mango aptamer sequence we can dramatically increase the sensitivity of NASBA to 1.5 RNA molecules per microliter. As this isothermal nucleic acid detection scheme directly produces a fluorescent reporter, real-time detection is intrinsic to the assay. Nested Mango NASBA is highly specific and, in contrast to existing RNA detection systems, offers a cheap, simple, and specific way to rapidly detect single-molecule amounts of pathogenic RNA.

Highly specific, multiplexed isothermal pathogen detection with fluorescent aptamer readout

“…However, as others have observed in NASBA reactions, untemplated amplification occurred in templatefree controls (Figures 2A-2E). 8 We speculated that unpromoted T7 RNA polymerase activity, as was recently reported, 10 could have been the source of the observed false positives. Given that either the sense or antisense RNA strand is sufficient to seed a NASBA reaction, and that our NASBA primers contain the T7 RNA polymerase promoter in one primer and the aptamer coding sequence in the other, a small amount of primer dimer could cause a false positive.…”

“…Furthermore, unlike dsDNA, RNA can exhibit diverse functional behaviors, including binding of fluorogenic dyes and enzymatic activity, enabling a wide range of potential readouts. 8 A common pitfall associated with isothermal amplification reactions is nonspecific amplification, which cannot be mitigated by thermal cycling, as in PCR. Investigators have employed a range of techniques to mitigate this pitfall, including engineered thermally stabilized polymerases and two-step nested reactions targeting different regions of the amplicon of interest (Nested Mango NASBA/NMN).…”

“…These strategies, while they enable high sensitivity and specificity, necessarily add complexity to the reaction. 8,9 One potential source of nonspecific amplification in reactions employing viral polymerases, such as T7 RNA polymerase, is unpromoted transcription. This latent behavior of T7 RNA polymerase was recently shown to be the reason transcripts produced by this enzyme can exhibit innate immune activation.…”

Isothermal SARS-CoV-2 Diagnostics: Tools for Enabling Distributed Pandemic Testing as a Means of Supporting Safe Reopenings

“…used a competitor DNA duplex to do so. 79 , 81 As with other multienzyme isothermal reactions, the use of multiple enzymes necessarily adds complexity to manufacturing processes.…”

Developments in integrating nucleic acid isothermal amplification and detection systems for point-of-care diagnostics

“…Similarly, the Unrau laboratory developed Nested Mango NASBA that featured an outer primer NASBA reaction where amplification of target analytes and primer artifacts occurred, followed by a second inner Mango NASBA reaction where primers containing a Mango III tag were added to specifically bind to the products of interest to generate fluorescent signals ( Abdolahzadeh et al, 2019 ). This system allowed them to reduce amplification of primer artifacts, resulting in greater sensitivity and specificity, and allowed them to achieve LODs of 25 pM for E. coli ClpB RNA and 2.5 aM for the Pseudomonas fluorescens ClpB target.…”