Nucleic acid extraction-free isothermal amplification based on magnetic beads capture and ligation

Abstract

Objective Capture and ligation probe-loop-mediated isothermal amplification CLIP-LAMP method with-out nucleic acid extraction based on magnetic beads was established to provide a new technique for the detection of human immunodeficiency virus (HIV) in large volume samples. Methods Our team earched a conservative sequence of the virus and used specific soft-ware to design a corresponding capture probe and detection probe. Streptavidin-coated magnetic beads was bound to biotin-labeled DAN molecules, connecting capture probe. The nucleic acid extraction was deleted because the capture probe was able to bind target DNA or RNA directly and specifically. After the ligation step, the detection probe was used as the final template to participate in the fluorescence quantitative loop-mediated isothermal amplification reaction.This method was used for the detection of HIV in vitro transcription (IVT) to assess the sensitivity and specificity of the method. Results This method was successfully applied to the detection of HIV IVT with the detection rate of 32 CPs/μL. The method was capable of processing 1 ml of samples, and the detection was completed in two hours. Conclusions The nucleic acid extraction-free loop-mediated isothermal amplification based on magnetic beads capture provides a new method and for rapid and simple detection of HIV and potentially other pathogenic microbes in large volume samples.

Keywords

• magnetic bead,hiv,loop-mediated isothermal amplification