The single-step multiplex reverse transcription- polymerase chain reaction assay for detecting H5 and H7 avian influenza A viruses.
Avian influenza (AI) A virus subtypes H5 and H7 cause severe disease in domestic poultry, including chickens and turkeys. Moreover, H5 and H7 AI A viruses can cross the species barrier from poultry to humans. In the present study, we have developed a single-step multiplex reverse transcription-polymerase chain reaction assay (RT-PCR) for detecting H5 and H7 AI A viruses. This assay was applied to the poultry isolates with the aim of establishing a surveillance method to monitor possible transmission to humans. Two subtype-specific primer sets capable of producing PCR products of 157 and 326 base pairs corresponding to AI A virus H5 and H7 subtypes, respectively, were utilized in a one-step and one-tube reaction. The single-step multiplex RT-PCR assay developed in this study was found to be specific for detecting H5 and H7 AI A viruses. No specific amplification bands were detected with total nucleic acids extracted from other influenza hemagglutinin subtypes and other viral pathogens. The sensitivity of this assay was about 10(3) RNA copies/microl. In conclusion, this novel single-step multiplex RT-PCR is a simple assay with high potential for rapid, specific and cost effective laboratory diagnosis of H5 and H7 AI A virus isolates from clinical specimens of poultry.
Thontiravong
Aunyaratana
A
Faculty of Veterinary Science, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand.
Payungporn
Sunchai
S
Keawcharoen
Juthatip
J
Chutinimitkul
Salin
S
Wattanodorn
Sumitra
S
Damrongwatanapokin
Sudarat
S
Chaisingh
Arunee
A
Theamboonlers
Apiradee
A
Poovorawan
Yong
Y
Oraveerakul
Kanisak
K
D000818:Animals; D001717:Birds / Q000821:virology; D004587:Electrophoresis, Agar Gel; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000302:isolation & purification; D053126:Influenza A Virus, H7N7 Subtype / Q000235:genetics / Q000302:isolation & purification; D009980:Influenza A virus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D005585:Influenza in Birds / Q000175:diagnosis / Q000821:virology*; D012367:RNA, Viral / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D012367:RNA, Viral