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Volume 47, Number 3, June 2014

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 


Kuo-Chien Tsao, Chiuan-Chian Chiou, Tai-Long Chen, Chung-Guei Huang, Erh-Fang Hsieh, Shin-Ru Shih


Received: April 6, 2012    Revised: July 17, 2012    Accepted: September 24, 2012   

 

Corresponding author:

Corresponding author. Research Center for Emerging Viral Infections, Chang Gung University, 259, Wen-Hua 1st Road, Kwei-Shan, Taoyuan 333, Taiwan.
Correspondence on virology and other aspects: srshih@mail.cgu.edu.tw. 



 

Background and purpose: 

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 × 104 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. 



 

Key words:

Drug resistance, Influenza virus, Peptic nucleic acid based PCR