Colorimetric detection of African swine fever (ASF)-associated microRNA based on rolling circle amplification and salt-induced gold nanoparticle aggregation

Abstract

African swine fever (ASF) is a severe viral disease with a high mortality rate in domestic and wild pigs, for which no effective vaccine and antiviral drugs are available. The great infectivity of the ASF virus highlights the need for sensitive, simple, and on-site detection assays of ASF. We herein developed a colorimetric sensing strategy for the detection of an ASF-associated miRNA, based on isothermal rolling circle amplification (RCA) and salt-induced gold nanoparticle aggregation. Ssc-miR-451 was selected as the target ASF biomarker due to its high expression in ASF virus-infected pigs. With a red-purple-blue color shifting, this biosensing platform offers convenient detection of ssc-miR-451 with a UV–Vis spectrometer or the naked eye. The proposed assay exhibits a dose-response relationship between the optical absorbance ratio (A525/A640) and the amounts of ssc-miR-451, with a detection limit calculated as 3.56 fmol (equivalent to 11.86 pM in 300 μL reaction mixture). This assay's coefficient of variation (CV%) was determined to be less than 5.95%, revealing its reproducibility is satisfactory. In addition, the newly developed method was successfully applied in the detection of spiked ssc-miR-451 in pig serum samples. In light of its simplicity, convenience (colorimetric), sensitivity, and energy efficiency (isothermal amplification), this biosensing strategy presents great potential to be applied in the local swine industry and pig farming for screening of viral diseases affecting pigs.