Single step multiplex real-time RT-PCR for H5N1 influenza A virus detection.
H5N1 influenza A virus causes a rapidly fatal systemic disease in domestic poultry and spreads directly from poultry to mammalian species such as leopards, tigers and humans. The aim of this study was to develop a multiplex real-time RT-PCR for rapid detection of H5N1 influenza A virus. The selected primers and various labeled TaqMan MGB reporter probes corresponding to M, H5 and N1 were used in a single step multiplex real-time RT-PCR to simultaneously detect triple fluorescent signals. In order to validate the method, 75 clinical specimens infected with H5N1 isolated from both poultry and mammals, as well as various specimens of other subtypes and RNA from other viral pathogens of poultry and human were tested. The results showed that the multiplex real-time RT-PCR assays can be applied to detect virus suspensions of H5N1 influenza A virus from a wide host range and demonstrated the sensitivity of the assay amounted to approximately 10(2)-10(3)copies/mul. In conclusion, the highlights of this particular method lie in its rapidity, specificity and sensitivity thus rendering it feasible and effective for large-scale screening at times of H5N1 influenza A virus outbreaks.
Payungporn
Sunchai
S
Center of Excellence in Viral Hepatitis Research, Faculty of Medicine, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand.
Chutinimitkul
Salin
S
Chaisingh
Arunee
A
Damrongwantanapokin
Sudarat
S
Buranathai
Chantanee
C
Amonsin
Alongkorn
A
Theamboonlers
Apiradee
A
Poovorawan
Yong
Y
D000818:Animals; D001717:Birds; D017931:DNA Primers; D005453:Fluorescence; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000821:virology*; D007251:Influenza, Human / Q000821:virology*; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000235:genetics; D009976:Orthomyxoviridae Infections / Q000662:veterinary* / Q000821:virology; D046009:Panthera; D011200:Poultry; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014763:Viral Matrix Proteins / Q000235:genetics
D017931:DNA Primers; D019267:Hemagglutinin Glycoproteins, Influenza Virus; C488936:M1 protein, Influenza A virus; D014763:Viral Matrix Proteins; D009439:Neuraminidase