Polymerase chain displacement reaction

BioTechniques

Claire L. Harris¹  Guoliang Fu¹  Luke Alphey¹  Ken E. Olson²  Irma J. Sanchez-Vargas² 

[1] 1Oxitec Ltd, Abingdon, Oxfordshire, UK;4Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, USA;

关键词: quantitative PCR;    real-time PCR;    virus;    diagnostics;    nested primers;    dengue;   

DOI  :  10.2144/000113951

来源: DOAJ

【 摘 要 】

Quantitative PCR assays are now the standard method for viral diagnostics. These assays must be specific, as well as sensitive, to detect the potentially low starting copy number of viral genomic material. We describe a new technique, polymerase chain displacement reaction (PCDR), which uses multiple nested primers in a rapid, capped, one-tube reaction that increases the sensitivity of normal quantitative PCR (qPCR) assays. Sensitivity was increased by approximately 10-fold in a proof-of-principle test on dengue virus sequence. In PCDR, when extension occurs from the outer primer, it displaces the extension strand produced from the inner primer by utilizing a polymerase that has strand displacement activity. This allows a greater than 2-fold increase of amplification product for each amplification cycle and therefore increased sensitivity and speed over conventional PCR. Increased sensitivity in PCDR would be useful in nucleic acid detection for viral diagnostics.

【 授权许可】

Unknown