Detection of low copies of drug-resistant influenza viral gene by a single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe.

Influenza virus infection causes endemics almost yearly and pandemics occasionally. Although antivirals are available for the clinical treatment of influenza virus infection, the emergence of a drug-resistant virus has reduced the effectiveness of therapy and prophylaxis. Therefore, the timely detection of drug-resistant influenza viruses is important. A single-tube reaction using peptide nucleic acid (PNA) as both a polymerase chain reaction (PCR) clamp and a sensor probe was established to detect the low numbers of copies of viral genes that carry the resistant marker. Influenza A H1N1 viruses resistant to a clinically used antiviral, amantadine, are selected for the experimental design. The PNA-mediated reverse transcription-PCR detected 10 copies/μL of RNA from the resistant strain among 2 × 10(4) copies/μL of RNA from the sensitive strain. A rapid and sensitive method was established for detecting low numbers of drug-resistant genes of the influenza virus. The assay would help to monitorthe emergence of adrug-resistant influenza virus.