A real-time RT-PCR for detection of clade 1 and 2 H5N1 influenza A virus using locked nucleic acid (LNA) TaqMan probes.
BACKGROUND
The emergence and co-circulation of two different clades (clade 1 and 2) of H5N1 influenza viruses in Vietnam necessitates the availability of a diagnostic assay that can detect both variants.


RESULTS
We developed a single real-time RT-PCR assay for detection of both clades of H5N1 viruses, directly from clinical specimens, using locked nucleic acid TaqMan probes. Primers and probe used in this assay were designed based on a highly conserved region in the HA gene of H5N1 viruses. The analytical sensitivity of the assay was < 0.5 PFU and 10-100 ssDNA plasmid copies. A total of 106 clinical samples (58 from patients infected with clade 1, 2.1 or 2.3 H5N1 viruses and 48 from uninfected or seasonal influenza A virus-infected individuals) were tested by the assay. The assay showed 97% concordance with initial diagnostics for H5 influenza virus infection with a specificity of 100%.


CONCLUSIONS
This assay is a useful tool for diagnosis of H5N1 virus infections in regions where different genetic clades are co-circulating.
Thanh
Tran Tan
TT
Oxford University Clinical Research Unit, Hospital for Tropical Diseases, 190 Ben Ham Tu, Dist 05, Ho Chi Minh City, Viet Nam. thanhtt@oucru.org
Pawestri
Hana Apsari
HA
Ngoc
Nghiem My
NM
Hien
Vo Minh
VM
Syahrial
Harun
H
Trung
Nguyen Vu
NV
van Doorn
Rogier H
RH
Wertheim
Heiman F L
HF
Chau
Nguyen Van Vinh
NV
Ha do
Quang
Q
Farrar
Jeremy J
JJ
Hien
Tran Tinh
TT
Sedyaningsih
Endang R
ER
de Jong
Menno D
MD
D017124:Conserved Sequence; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D015345:Oligonucleotide Probes / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014744:Vietnam
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; C109963:hemagglutinin, avian influenza A virus; C477371:locked nucleic acid