Nucleic acid based probes have high selectivity for single nucleotide mutations and are therefore useful for detecting sequence variances in biological samples for point-of-care (POC) diagnostics. However, sample amplification prior to analysis is often required. The Nucleic Acid Sequence Based Amplification (NASBA) technique is promising for nucleic acid based POC diagnostics. This is due to the isothermal reaction conditions, the possibility to use lyophilized enzymes to avoid the need for cold storage, and the fact that the product is single stranded RNA. The RNA product is beneficial because its low chemical stability can reduce cross contamination during assays. At the same time, being single-stranded, RNA can be easily interrogated by hybridization probes during the downstream amplicon detection step. In most assays, the amplicon undergoes end-point size- or sequence-based analysis to determine if amplification was successful. This adds to the complexity of the assay, which is undesirable for a POC test. An amplicon-specific probe that can monitor the progress of the amplicon synthesis in real time is advantageous, since the results can be obtained sooner with the decreased probability of cross-contamination.<br/>The ssRNA product of the NASBA reaction makes this amplification method ideal for real time diagnostics, since there is limited competition between the sensors and the compliment strand. Currently, a commercially available kit for real-time NASBA (e.g. from Life Sciences Technologies, Inc.) employs a sequence-specific molecular beacon (MB) probe. MB probes are known to suffer from high background in complex protein-containing mixtures, since they can interact with and be opened by the proteins, which results in high fluorescence of the probe even without a nucleic acid target present. Such limitation compromises the detection limits of MB-based real-time NASBA assays and would require more sophisticated and expensive equipment for noise suppression (e.g. qPCR instruments).<br/>We propose to advance the usability of real-time NASBA detection of viral infections by utilizing split approach to design hybridization probes for real-time monitoring of RNA amplicon. In this work we demonstrate the use of a split aptamer fluorescent dye based probe and split deoxyribozyme fluorescent probe for real-time detection of flaviviruses in a NASBA reaction. This approach is also applicable for real-time monitoring and kinetic studies of in-vitro transcription.

Real time monitoring of NASBA reaction using split hybridization probes

2019 Spring National Meeting

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