Qualitative detection of avian influenza A (H5N1) viruses: a comparative evaluation of four real-time nucleic acid amplification methods.
The aim of this study was to determine the performance of real-time amplification based methods - NASBA, TaqMan, RT-FRET, and RT-PCR LUXtrade mark formats - for the detection of influenza A (H5N1) virus RNA. In an analysis of 54 samples obtained from a range of animal species in Thailand during the period 2003-2006, results showed that the NASBA (H5=98.2%, N1=96.3%), TaqMan (H5=98.2%, N1=96.3%) and FRET (H5=98.2%, N1=96.3%) had significantly higher rates of positive detection than LUX (H5=94.4%, N1=50.0%; P<0.001) for influenza A, H5 and N1 isolates. There were no false-positive results from any methods used in the negative-control group of samples. The limits of analytical detection were at least 10copies/reaction in real-time NASBA and LUX assays, while FRET and TaqMan assay appeared to be less sensitive at > or =100copies/reaction. The assays were relatively specific without cross-reactivity to a number of other influenza strains or viral pathogens. In conclusion, our study demonstrated that real-time NASBA, TaqMan and FRET assays can be used to detect influenza A (H5N1) from a wide range of hosts, and be specific for H5N1 samples obtained during different outbreaks (2003-2006). All assays provided the benefit of rapid influenza H5N1 identification for early diagnosis, in the range of hours, and they are well suited to high throughput analyses.
Chantratita
Wasun
W
Virology and Molecular Microbiology Unit, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. rawct@mahidol.ac.th
Sukasem
Chonlaphat
C
Kaewpongsri
Supaporn
S
Srichunrusami
Chutatip
C
Pairoj
Wantanit
W
Thitithanyanont
Arunee
A
Chaichoune
Kridsada
K
Ratanakron
Parntep
P
Songserm
Thaweesak
T
Damrongwatanapokin
Sudarat
S
Landt
Olfert
O
D000818:Animals; D001717:Birds / Q000382:microbiology; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics*; D005585:Influenza in Birds / Q000453:epidemiology / Q000382:microbiology; D007251:Influenza, Human / Q000453:epidemiology / Q000382:microbiology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013785:Thailand