Scanometric Detection of Tomato Leaf Curl New Delhi Viral DNA Using Mono- and Bifunctional AuNP-Conjugated Oligonucleotide Probes

Abstract

Scanometric detection of tomato leaf curl New Delhi viral DNA using AuNP-conjugated mono- and bifunctional oligo probes through direct DNA hybridization assay (DDH assay) and sandwich DNA hybridization assay (SDH assay) with silver enhancement was developed. Tomato leaf curl New Delhi virus (ToLCNDV) coat protein gene-specific thiol-modified ssoligo probes were used for the preparation of mono- and bifunctional AuNP-ssoligo probe conjugates (signal probes). ssDNA arrays were prepared using polymerase chain reaction (PCR), rolling circle amplification (RCA), genomic DNAs fragments, and phosphate-modified positive control/capture probes through 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/1-methylimidazole conjugation on the amine-modified glass slide (GS) surface. In the DDH assay, signal probes were directly hybridized with ssDNA array of positive control and ToLCNDV DNA samples and the detection signals were amplified by silver enhancement. Dark black/gray colors were developed on the GS by the result of Ag enhancement, which can be visualized and discriminated by the naked eye. The images were captured using a simple flatbed scanner, and the determined amounts of signal probes were hybridized with their target DNA. Similarly, the SDH assay also performed through two rounds of hybridization between capture probes and target DNA; target DNA and signal probes followed by silver enhancement. The detection signals were found higher in the PCR sample than the RCA and genomic DNA samples because of the presence of increased copy numbers of complementary DNAs in PCR samples. Further, bifunctional AuNP-ssoligo probe shows higher intensity of detection signal than monofunctional probes because it can be hybridized with both strands of dsDNA targets. Moreover, the DDH-based scanometric method showed higher detection sensitivity than the SDH assay-based scanometric method. Overall, bifunctional signal probes showed more detection sensitivity than monofunctional probes in scanometric methods based on both DDH and SDH assays. The limit of detection of this developed scanometric method was optimized (100 zM to 100 pM concentration). Further, DDH assay-based scanometric method shows significant advantages over the SDH assay method, such as cost-effectiveness, because it requires only single probes (signal probes), less time-consuming by the need of only single-step hybridization, and higher detection sensitivity (up to zM). To the best of our knowledge, this is the first attempt made to develop a scanometric-based nanoassay method for the detection of plant viral DNA. This approach will be a remarkable milestone for the application of nanotechnology in the development of nanobiosensor for plant pathogen detection.

Molecular Approaches for Low-Cost Point-of-Care Pathogen Detection in Agriculture and Forestry

“…Another colorimetric method proposed for application in POC plant pathogen detection exploits the formation of a large amount of pyrophosphate ions as a byproduct of LAMP reactions (Chang et al, 2013;Lin et al, 2015). Several other methods have been exploited to perform POC colorimetric detection of nucleic acids for plant or human pathogen detection, as for example, the use of hydroxyl naphthol blue (HNB) (Harper et al, 2010), the activation of peroxidase-like deoxyribozyme (PDz) (Reed et al, 2019), pH-sensitive dyes (Hamidi and Perreault, 2019), and silver enhancement (Dharanivasan et al, 2019). Despite many advantages, the colorimetric detection methods still present some drawbacks, mainly the lack of sensitivity and specificity.…”

Ultrasensitive nano-gold labelled, duplex lateral flow immunochromatographic assay for early detection of sugarcane mosaic viruses

“…The comparative sensitivity of one-week-old LFIA strips imbedded with the conjugates of Citrate-AuNPs and Cysteamine-AuNPs was tested using two-fold diluted purified SCSMV, and the intensity of test and control line images were recorded. Citrate-AuNPs synthesized by standard procedure and antibodies were conjugated followed by the protocol described elsewhere 27,45 .…”

Signal Transduction Strategies for Analyte Detection Using DNA‐Based Nanostructures

“…Scanometric assays represent one of the earliest reported detection techniques that rely on DNA‐based nanostructures. Introduced in 2000 by Mirkin and co‐workers, these assays offer a relatively simple and rapid method for the detection of nucleic acid targets down to concentrations of about 100 z m [7, 27, 28] …”