Visual detection of Maize chlorotic mottle virus by asymmetric polymerase chain reaction with unmodified gold nanoparticles as the colorimetric probe

Abstract

Maize chlorotic mottle virus causes corn lethal necrosis disease and can transmit via infected maize seeds. It remains a challenge to detect this virus to prevent its introduction and infection and spread transmission fields. For this purpose, visual detection of Maize chlorotic mottle virus was investigated in cooperation with a asymmetric polymerase chain reaction and unmodified gold nanoparticles (AuNPs). Detection relies on the fact that the dispersed AuNPs solution is red due to the intense surface plasmon absorption band at 520 nm. The color changed to grey after saline induction; single-stranded DNA (ssDNA) electrostatically adsorbed onto naked AuNPs stabilizes them against salt-induced aggregation, whereas the AuNPs solution in the presence of ssDNA of asymmetric RT-PCR target products of Maize chlorotic mottle virus remains red even after saline induction. A remarkable color change from red to grey immediately occurred in absence of ssDNA amplified from Maize chlorotic mottle virus with asymmetric RT-PCR. After optimization of the asymmetric RT-PCR primer ratios, concentration of NaCl, and evaluation of specificity and sensitivity of a novel assay, visual detection of Maize chlorotic mottle virus using unmodified AuNPs and asymmetric RT-PCR has been developed with simple preparation of samples in our study. Through this assay, as low as 23.9 pg μL⁻¹ of RNA of Maize chlorotic mottle virus was detected visually by the naked eye without the need for any sophisticated, expensive instrumentation and biochemical reagents. The results indicate that this assay is highly species-specific, simple, low-cost, and visible for easy detection of Maize chlorotic mottle virus in plant tissues. Therefore, visual detection of Maize chlorotic mottle virus is a potentially useful tool for middle- or small-scale corporations and entry–exit inspection and quarantine bureaux to detect Maize chlorotic mottle virus in maize seeds or plant tissues.