Multiplex Detection of RNA Viruses Based on Ligation Reaction and Universal PCR Amplification

To detect several RNA viruses simultaneously, a method based on multiplex ligation reaction combined with multiplex qPCR or multiplex PCR+capillary electrophoresis was established to detect four RNA viruses: human immunodeficiency virus (HIV), hepatitis C (HCV), influenza A virus (IAV) H1N1 and H5N1. The experimental conditions including ligation probe concentration, annealing procedure, ligation temperature and ligase dosage were optimized intensively. We found that the specificity of the ligation reaction was affected by the probe concentration predominantly, high-probe concentration (100 nM) resulted in splint-independent ligation with efficiency comparable to that with RNA splint. The sensitivity of the ligation reaction was affected by the annealing mode apparently as the sensitivity of the step-down annealing mode was 100 times higher than that of the isothermal annealing at 37 °C. Under the optimized condition, this assay could detect virus RNA as low as 16 viral copies per reaction in doubleplex and triplex real-time quantitative PCR detection with satisfactory specificity and precision. By multiplex PCR+capillary electrophoresis, four RNA viruses could be detected in one tube with the sensitivity of 10 copies per reaction.