Extremely sensitive, background-free gene detection using binary probes and beta replicase.

Abstract

We have developed a specific and sensitive nucleic acid amplification assay that is suitable for routine gene detection. The assay is based on a novel molecular genetic strategy in which two different RNA probes are hybridized to adjacent positions on a target nucleic acid and then ligated to form an amplifiable reporter RNA. The reporter RNA is then replicated up to a hundred billion-fold in a 30-min isothermal reaction that signals the presence of the target. The assay can detect fewer than 100 nucleic acid molecules; it provides quantitative results over a wide range oftarget concentrations and it employs a universal format that can detect any infectious agent.

Genomics, Molecular Epidemiology and Diagnostics of Infectious hypodermal and hematopoietic necrosis virus

“…By using specific primer pairs, the closed circular C probe is amplified under isothermal condition. After its initial discovery [98], it became widely used for diagnosis and single nucleotide polymorphism analysis [5,53,73,87,88,91]. In 2006, a simple and sensitive RAM assay was developed for detection of IHHNV by Teng et al [86].…”

Replicable and recombinogenic RNAs

“…The background RNA synthesis caused by non-specifically bound probes was eliminated by introducing compound binary probes consisting of two fragments capable of producing the full-sized replicable RNA when ligated upon hybridization next to one another on a target molecule if this is present in the analyzed sample [28]. However, this approach has not become a routine assay because even short inserts often inhibit replication of RQ RNA vectors, and because binary RNA probes can self-recombine to produce replicable RNAs in the absence of any target or ligase (see below).…”

Detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Litopenaeus vannamei by ramification amplification assay

“…Under isothermal condition, the circular C probe is subsequently amplified with a pair of C probe-specific primers, driven by Bst DNA polymerase with high strand-displacement activity. This amplification is also named hyperbranched rolling circle amplification or cascade rolling circle amplification.After RAM was first demonstrated by Zhang et al (1998), it became widely applied to DNA-, RNA-or protein-based diagnosis and single nucleotide polymorphism analysis (Barany 1991, Tyagi et al 1996, Lizardi et al 1998, Thomas et al 1999. Other isothermal amplification methods, such as loop-mediated isothermal amplification (LAMP), have also been reported to detect targets at the fentogram range under isothermal conditions (Kono et al 2004).…”