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 pathogen-related 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.

Keywords: Mango; RNA; TO1 fluorophore; isothermal amplification; nested NASBA; nucleic acid detection; single-molecule detection.

FIGURE 1.

Insertion of RNA Mango aptamers into RNA NASBA. (A) Traditional NASBA uses two primers to produce an RNA product. Artifacts are also commonly produced when amplified by primers PA and PB and are indicated by black and gray products. (B) Mango NASBA system features the addition of a Mango aptamer template sequence on PB primer, resulting in the production of an RNA product containing a fluorescent Mango tag after T7 transcription, also sensitive to artifacts. (C) Nested Mango NASBA features an outer primer NASBA reaction whose products are then diluted and fed into a second inner Mango NASBA reaction, reducing artifactual amplification of products.

FIGURE 2.

Nested Mango NASBA is sensitive and specific to target RNA sequence and is robust even when an unrelated nucleic acid sample is added. (A) Unnested outer (E. coli ClpB RNA, E. coli primers P1/P2A) sensitivity. (B) Nested RNA Mango dramatically improves sensitivity. E. coli primers (P1/P2B and P3/P4) with E. coli ClpB RNA (Ec/Ec) and is shown in blue. Using the same E. coli primers, P. fluorescens ClpB RNA was added (Ec/Pf) instead of E. coli; target is shown in purple. P. fluorescens primers (P5/P6 and P7/P8) with P. fluorescens ClpB RNA (Pf/Pf) is shown in red. (C) Nested Mango NASBA using E. coli primers as in panel B was performed with the inner NASBA time courses shown. (Blue) 150 E. coli target molecules/µL, (red) 150 E. coli target molecules/µL in the presence of 5 ng/µL of A549 Human Lung Carcinoma total nucleic acid, (black) no template added, (gray) 5 ng/µL of A549 Human Lung Carcinoma cell total nucleic acid. Error bars reflect the standard deviation of three replicates for all panels.