Self-primed isothermal amplification for genomic DNA detection of human papillomavirus

Rolling circle amplification (RCA) is an isothermal amplification technique with high efficiency and perfect accuracy for nucleic acids detection. However, RCA technique suffers the limitation to detect short DNA or RNA molecules. For long nucleic acid molecules, enzymatic restriction as well as heat denaturation process is usually required, which makes the amplification not effective and strictly isothermal. In this article, a simple and efficient one-pot self-primed isothermal amplification (SIA) was developed for detection of genomic DNA directly based on the combination of nicking endonuclease assisted strand displacement amplification (SDA) and exponential RCA. In virtue of numerous nicking sites on the genome, a pre-amplification of the whole genome was performed through SDA with the specific cleaving of nicking endonuclease. Meanwhile, the single strand DNA with HPV target sequence generated from SDA could hybrid with the circle probe as a primer and trigger the exponential RCA as a result of the existence of nicking endonuclease. As the reaction temperature and enzyme were the same, the amplification could be operated in one pot. The reaction solution after amplification was added on the electrode for hybridization with the sulfydryl probe to achieve the electrochemical signal. Based on the isothermal amplification, genotyping of HPV 11, 16, 18 and the detection of HPV 18 in Hela cell line were attempted with satisfied results. This approach should be a promising tool for pathogene detection in clinical diagnostics and research.