Detection of reverse transcription-PCR products by a simple and rapid light scattering technique

Abstract

A fast, sensitive and simple light scattering approach is developed to detect reverse transcription-PCR (RT-PCR) products. In the solution of HClO₄, the RT-PCR products can be denatured and aggregated to form large particles, which can result in very strong light scattering. The RT-PCR products of D1/D2 domain in yeast 26S rRNA are successfully quantified with the proposed method. The light scattering intensity is well proportional to the concentration of RT-PCR products in the range of 0.01–0.5 μg ml⁻¹ and 0.5–4.0 μg ml⁻¹, respectively. The light scattering method gives more sensitive results, typically, two orders of magnitude better than agarose gel electrophoresis with ethidium bromide staining. The novel method has many advantages over conventional gel-based methods and other non-gel-based methods—fast detection within 5 min and stable signal within 60 min, a simple detection system including only one cheap chemical agent (HClO₄) and direct one-step detection without purification of the PCR reaction products—showing great potential in nucleic acid-based clinical diagnostics and other related fields.