In situ strand displacement amplification: an improved technique for the detection of low copy nucleic acids.

Abstract

Strand displacement amplification (SDA) is an isothermal, enzymatic method for the exponential synthesis of target nucleic acids which traditionally requires two pairs of oligoprimers. One pair, described as "bumpers", serves to generate target specific single stranded DNA after heat denaturation of double stranded DNA subsequent to primer annealing and extension. We report here the in situ (IS) application of SDA (IS-SDA) using nicked DNA targets which eliminate the need for heat denaturation and bumpers. Productive nicking can be generated by restriction endonuclease activity, exposure to dry heat, paraffin embedding, or mild depurination. IS-SDA, followed by in situ hybridization (ISH) allows for the routine detection of single copy HIV-1 DNA as well as HPV DNA in both cell lines and tissue sections. Detection of hepatitis C RNA in liver biopsy specimens using reverse transcription IS-SDA (RT-IS-SDA) was performed with direct incorporation of the reporter nucleotide. The amplification of RNA targets does require bumper primers, although not an initial denaturation step. Extensive analyzes demonstrated that SDA-ISH is as sensitive as polymerase chain reaction (PCR)-ISH and that each are more sensitive than in situ hybridization coupled to catalyzed signal amplification (CSA). Moreover. SDA-ISH is technically much simpler than PCR-ISH because it is isothermal and does not require an initial denaturation.