Detection of Two Orchid Viruses Using Quartz Crystal Microbalance-Based DNA Biosensors

Abstract

We have developed a piezoelectric DNA-sensor based on DNA-RNA hybridization for the detection of two orchid viruses, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). Specific oligonucleotide probes modified with a mercaptohexyl group at the 5'-phosphate end were directly immobilized onto 10-MHz AT-cut quartz crystal microbalance (QCM). QCMs coated with such oligonucleotide probes were exposed to test solutions containing viral RNA for hybridization. Various experimental conditions evaluated were (i) DNA probe coating concentration, (ii) sensitivity and specificity of the probes at different hybridization temperatures, and (iii) effects of incubation temperature on the hybridization time. The specific nucleotide probe-coated QCM-based DNA sensors were able to detect both CymMV and ORSV in quantities as low as approximately 1 ng in purified RNA preparations and 10 ng in the crude sap of infected orchids. This is the first application of a DNA biosensor for the detection of plant viruses.

Gravimetric Viral Diagnostics: QCM Based Biosensors for Early Detection of Viruses

“…Hereby, some of the most promising results of DNA-QCM biosensors for different viruses are discussed. For instance, DNA-QCM biosensors have been successfully tested for the detection of hepatitis B virus (HepBV) [105][106][107], hepatitis C virus (HepCV) [117], vaccinia virus [108], viral haemorrhagic septicaemia virus (VHSV) [109], human papilloma virus (HPV) [110], dengue virus [118], and orchid viruses such as cymbidium mosaic virus (CymMV) and odontoglossum ringspot virus (ORSV) [111].…”

Discovering Fragrance Biosynthesis Genes from Vanda Mimi Palmer Using the Expressed Sequence Tag (EST) Approach

“…So far, the identification of CymMV infection is done through serological, bioassay or electron microscopy. Those techniques include enzyme-linked immunosorbent assays (ELISA), dot-blot immunoassay (DBIA), rapid immunofilter paper assay (RIPA), immunosorbent electron microscope (ISEM), DIG-labelled cRNA probes, reverse transcription polymerase chain reaction (RT-PCR), quartz crystal microbalance-based DNA biosensors and TaqMan real-time quantitative RT-PCR (Eun et al, 2002;Eun & Wong, 2000;Hsu et al, 1992;Hu et al, 1998;Khentry et al, 2006;Rani et al, 2010).…”

A survey of the 2001 to 2005 quartz crystal microbalance biosensor literature: applications of acoustic physics to the analysis of biomolecular interactions

“…This detection limit was again comparable to an ELISA, but as before considerably faster. This group also developed an assay to genotype the same viruses using specific oligonucleotide probes in place of the antibodies, with similar detection limits (Eun et al, 2002a). Similarly, an assay for molecular diagnosis of hepatitis B virus infection showed a dynamic range of 0.02-0.14 mg ml À1 of nucleic acid, an amount requiring prior PCR amplification of the sample .…”