Real-time multiplex PCR for rapid detection of enteroviruses, adenoviruses and hepatitis A virus in clinical specimens

Real-time multiplex polymerase chain reaction (PCR) with internal positive control (IPC) was developed for simultaneous detection of adenoviruses (AV), enteroviruses (EV) and hepatitis A virus (HAV). Primes and probes labeled with different fluorophores (FAM, R6G, ROX, and Cy5) and able to detect up to four viral RNAs (DNAs) with high specificity in a single tube in real-time PCR were designed. Sensitivity and specificity of the method was estimated using cultural strains of 8 serotypes of EV, 5 serotypes of AV and 2 clinical specimens containing HAV. Sensitivity of the method for detection of polioviruses types 1, 2, and 3 (Sabin vaccine strains) was 0.5--1 TCID50 per reaction mixture. Thirty clinical specimens were analyzed by the multiplex PCR with and without IPC, and by mono-specific PCR. Comparison of these methods with cultural one revealed results agreement in 86.7% in case of multiplex PCR with IPC and in 100% in case of multiplex PCR without IPC and mono-specific PCR. This method can be used for rapid diagnostics of enteric viral infections as well as for determination of viral contamination level of water. As intermediate results of the study the methods for quantitative assessment of HAV, AV, and EV nucleic acids were developed which are convenient tools for the control of antiviral therapy effectiveness.