Rapid detection of avian influenza virus a and subtype H5N1 by single step multiplex reverse transcription-polymerase chain reaction.
Outbreaks of H5N1 highly pathogenic avian influenza (HPAI) virus caused great economic losses to the poultry industry and resulted in human deaths in Thailand and Viet Nam in 2004. Rapid typing and subtyping of H5N1 viruses, especially from clinical specimens, are desirable for taking prompt control measures to prevent the spread of the disease. Here, we developed a set of oligonucleotide primers able to detect, type and subtype H5 and N1 influenza viruses in a single step multiplex reverse transcription-polymerase chain reaction (RT-PCR). RNA was extracted from allantoic fluid or from specimens with guanidinium isothiocyanate reagent. Reverse transcription and PCR were carried out with a mixture of primers specific for influenza viruses of type A, subtype H5 and N1 in a single reaction system under identical conditions. The amplified DNA fragments were analyzed by agarose gel electrophoresis. All the H5N1 viruses tested in the study and the experimental specimens presented three specific bands by the method established here. The results presented here suggest that the method described below is rapid and specific and, therefore, could be valuable in the rapid detection of H5N1 influenza viruses in clinics.
Wei
Hui-Ling
HL
Department of Infectious Disease, College of Veterinary Medicine, China Agricultural University, 100094 Beijing, People's Republic of China.
Bai
Gui-Rong
GR
Mweene
Aaron S
AS
Zhou
Ying-Chun
YC
Cong
Yan-Long
YL
Pu
Juan
J
Wang
Shuai
S
Kida
Hiroshi
H
Liu
Jin-Hua
JH
D000818:Animals; D002645:Chickens; D017931:DNA Primers; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000175:diagnosis / Q000821:virology*; D007251:Influenza, Human / Q000175:diagnosis / Q000821:virology*; D011200:Poultry; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
D017931:DNA Primers; D012367:RNA, Viral