Isothermal DNA Amplification with Gold Nanosphere-Based Visual Colorimetric Readout for Herpes Simplex Virus Detection

Abstract

There is a need for rapid nucleic acid amplification and detection technologies suitable for point-of-care settings. PCR-based molecular diagnostics, portable thermocyclers, and rapid microfluidic techniques are emerging technologies (1), but the use of isothermal nucleic acid amplification would make less demanding instrumentation feasible. Most isothermal methods reported to date(2)(3)(4)(5)(6)(7) require reaction times >30 min. In this study, we describe very rapid isothermal DNA amplification through EXPAR (exponential amplification reaction)(8)(9) coupled with visual colorimetric detection facilitated by aggregation of DNA-functionalized gold nanospheres(10)(11)(12) applied to a genomic sequence derived from herpes simplex virus (HSV)1. Rapid detection of HSV infections is important in HIV-positive and immunocompromised individuals (13)(14), pregnant women, and newborns(15). PCR-based assays are superior to viral cultures and immunoassays for the diagnosis of HSV infections from cerebrospinal fluid(16) and genital, oral, and topical swabs(17)(18)(19)(20). New rapid, low-cost molecular diagnostic tools can enable broad-based testing of at-risk populations, providing effective patient treatment and preventing further disease spread. EXPAR (8)(9) isothermally amplifies a short oligonucleotide trigger 106- to 109-fold in <10 min. EXPAR occurs at 55 °C, permitting activity and stability of the polymerase and nicking endonuclease involved in the reaction(9). Trigger X primes an amplification template containing 2 complimentary X′ sequences and enables generation of the nicking enzyme recognition and cleavage site (see Fig. S1 in the Data Supplement that accompanies the online version of this Abstracts of Oak Ridge Posters at http://www.clinchem.org/content/vol53/issue11). Trigger extension and single-strand nicking create another trigger oligonucleotide, which dissociates from the amplification template. Through repeating cycles of elongating and nicking, the trigger is linearly amplified. Newly formed triggers prime other …