Single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) for influenza A virus subtype H5N1 detection.
Influenza A virus subtype H5N1 causes a rapidly fatal systemic disease in domestic poultry and spreads directly from poultry to humans. The aim of this study was to develop a rapid, cost-saving and effective method for influenza A virus subtype H5N1 detection. The selected primer set was used in single-step RT-PCR for simultaneous detection in multiplex format of the 276-, 189-, and 131-bp fragments, corresponding to sequences specific for M, H5 and N1. The amplified DNA fragments were clearly separated by agarose gel electrophoresis. The sensitivity of this assay was about 10(3) copies/microL. Moreover, this method can be applied to detect not only avian but also human influenza A virus subtype H5N1. In conclusion, the highlights of this particular method are its rapidity and cost-effectiveness, thus rendering it feasible and attractive for large-scale screening at times of influenza A virus subtype H5N1 outbreak.
Payungporn
Sunchai
S
Center of Excellence in Viral Hepatitis Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
Phakdeewirot
Piraya
P
Chutinimitkul
Salin
S
Theamboonlers
Apiradee
A
Keawcharoen
Juthatip
J
Oraveerakul
Kanisak
K
Amonsin
Alongkorn
A
Poovorawan
Yong
Y
D000818:Animals; D001717:Birds / Q000821:virology; D002645:Chickens / Q000821:virology; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000175:diagnosis / Q000821:virology*; D007251:Influenza, Human / Q000175:diagnosis / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity