Molecular beacon probes combined with amplification by NASBA enable homogeneous, real-time detection of RNA

Abstract

Molecular beacon probes can be employed in a NASBA amplicon detection system to generate a specific fluorescent signal concomitantly with amplification. A molecular beacon, designed to hybridize within the target sequence, was introduced into NASBA reactions that amplify the genomic RNA of potato leafroll virus (PLRV). During amplification, the probe anneals to the antisense RNA amplicon generated by NASBA, producing a specific fluorescent signal that can be monitored in real-time. The assay is rapid, sensitive and specific. As RNA amplification and detection can be carried out in unopened vessels, it minimizes the risk of carry-over contaminations. Robustness has been verified on real-world samples. This homogeneous assay, called AmpliDet RNA, is a significant improvement over current detection methods for NASBA amplicons and is suitable for one-tube applications ranging from high-throughput diagnostics to in vivo studies of biological activities.

Betanodavirus of Marine and Freshwater Fish: Distribution, Genomic Organization, Diagnosis and Control Measures

“…The method amplifies a target-specific product through oligonucleotide primers and the co-ordinated activity of 3 enzymes: reverse transcriptase, RNase H, and T7 RNA polymerase. Real-time detection in NASBA is performed by applying molecular beacons, which are incorporated directly into amplification reactions [72]. The real-time NASBA procedure for the detection of betanodavirus has been developed and sensitivity of this assay was compared to a conventional single-tube RT-PCR assay [113].…”

Potentials and limitations of molecular diagnostic methods in food safety

“…Recently, fluorescently labelled probes and a fluorescence scanner are employed to follow the NASBA real-time amplification of a viral RNA genome [28].…”

Real-time quantification of RNA polymerase activity using a “broken beacon”

“…Opposite ends are labeled with a fluorophore and quencher, respectively. As shown in Figure 1C, the loop structure is complementary to the target so that when the beacon binds to the product, the fluorophore is separated from the quencher, and fluorescence increases in proportion to the amount of DNA produced [1,3,[6][7][8][9].…”

“…Nucleic acid sequence-based amplification (NASBA) synthesizes RNA in an isothermal manner. Molecular beacons are the primary detection system used in conjunction with NASBA for real-time examination with typical detection limits in the low nM range [7,10]. While molecular beacons have been used to detect RNA production in situ, they tend to be at least a factor of 4-10 more expensive than simple fluor-labeled oligos and typically require sophisticated software packages for appropriate design [11].…”