A rapid and high sensitivity RNA detection based on NASBA and G4-ThT fluorescent biosensor

In recent years, various newly emerged and re-emerged RNA viruses have seriously threatened the global public health. There is a pressing need for rapid and reliable nucleic acid-based assays for detecting viral RNA. Here, we successfully developed a highly sensitive, easy-to-operate G4-ThT-NASBA system to detect viral RNA that no need for labeled primers and probes. Next, we tested the system for detecting the Classical Swine Fever Virus (CSFV), an RNA virus that causes a highly contagious disease in domestic pigs and wild boar and easily causes huge economic losses. Results showed that the system, integrated the G4-ThT fluorescent biosensor and NASBA (Nuclear acid sequence-based amplification),is capable to detect as little as 2 copies/μL of viral RNA without interfering by other swine viral RNA. Moreover, we were able to detect CSFV RNA within 2 h in serum samples taken from the field in a real-time mode. These findings indicate that the G4-ThT-NASBA system is a rapid, high sensitivity and easy-to-operate technique for RNA detection. The method also has the real-time detection capability which may be easily integrated in a highly automated system such as microfluidic chips.