Rapid diagnosis of H5N1 avian influenza virus infection by newly developed influenza H5 hemagglutinin gene-specific loop-mediated isothermal amplification method.
Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) is a unique gene amplification method that can be completed within 35 min at 62.5 degrees C. In the present study, RT-LAMP was used to develop a rapid and sensitive laboratory diagnostic system for the H5N1 highly pathogenic avian influenza (HPAI). The sensitivity of the system was 0.1-0.01 plaque-forming units per reaction for HPAI-H5N1 viruses belonging to the genetically and antigenically distinct clade 1, represented by A/Vietnam/JP1203/2004, and clade 2, represented by A/Indonesia/JP283/2006. This RT-LAMP sensitivity is 10-fold higher than the sensitivity of standard one-step RT-PCR. By using viral RNAs extracted from avian influenza viruses of H1-H15 hemagglutinin (HA) subtypes and human pathogenic respiratory viruses, it was confirmed that the RT-LAMP system amplifies specifically RNA of the H5 subtype virus. The system detected H5-HA genes in throat swabs collected from humans as well as from wild birds. These results suggest that the present RT-LAMP system is a useful diagnostic tool for surveillance of recent outbreaks of the HPAI-H5N1 virus.
Imai
Masaki
M
Laboratory of Influenza Viruses, Department of Virology 3, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashi-Murayama, Tokyo 208-0011, Japan.
Ninomiya
Ai
A
Minekawa
Harumi
H
Notomi
Tsugunori
T
Ishizaki
Toru
T
Van Tu
Phan
P
Tien
Nguyen Thi Kim
NT
Tashiro
Masato
M
Odagiri
Takato
T
D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D000956:Antigens, Viral / Q000235:genetics; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D046368:Crows; D017931:DNA Primers / Q000235:genetics; D005814:Genes, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000175:diagnosis* / Q000821:virology; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D008875:Middle Aged; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity
D000956:Antigens, Viral; D017931:DNA Primers; D019267:Hemagglutinin Glycoproteins, Influenza Virus; C109963:hemagglutinin, avian influenza A virus