Direct multiplex reverse transcription-nested PCR detection of influenza viruses without RNA purification.
This paper describes the development a of direct multiplex reverse transcription-nested polymerase chain reaction (PCR) method, devised for simultaneous detection and typing of influenza viruses. This method combines the direct reverse transcription reaction without RNA purification with the enhancement of sensitivity and specificity of nested PCR. The method successfully detected three major human influenza viruses: influenza virus A subtype 1 (H1N1) and subtype 3 (H3N2), and influenza B virus (B). The minimum number of virus particles (pfu/ml) necessary for detection in spiked saliva samples was 200 (H1N1), 140 (H3N2), and 4.5 (B). The method's sensitivity and simplicity will be convenient for use in clinical laboratories for the detection and subtyping of influenza and possibly other RNA viruses.
Song
Man Ki
MK
Laboratory Science Division, International Vaccine Institute, Seoul 151-600, Korea.
Chang
Jun
J
Hong
Yeongjin
Y
Hong
Sunghoi
S
Kim
Suhng Wook
SW
D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D006358:Hot Temperature; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000302:isolation & purification; D053122:Influenza A Virus, H3N2 Subtype / Q000235:genetics / Q000302:isolation & purification; D009981:Influenza B virus / Q000235:genetics / Q000302:isolation & purification; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D025202:Molecular Diagnostic Techniques / Q000379:methods; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012463:Saliva / Q000821:virology; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D012367:RNA, Viral