Detection of type and subtypes of influenza virus by hybrid formation of FRET probe with amplified target DNA and melting temperature analysis.
Fluorescence resonance energy transfer probes were selected for three sets of reverse transcription -polymerase chain reaction (RT-PCR), each in duplex format to detect: (1). Influenza virus type A or B; (2). Neuraminidase subtypes N1 or N2; and (3). Hemagglutinin subtypes H1 or H3 using LightCycler Instrument. A pair of probes targeted a type or subtype specific RT-PCR amplified gene segment. The presence of a target in a set of amplification reaction was detected by increased fluorescence over background emitted from the appropriate reporter fluorophore on probe-target hybrid formation and by melting temperature (T(m)) analysis of probe-target hybrid. The T(m) of a probe-target in a duplex amplification was distinctly different from the other, and thus T(m) value allowed specific detection of a target. Amplified product in each set of amplification was also examined by conventional agarose gel electrophoresis. The sensitivities of detection by fluorescence signal analysis were equal or ten times better than that detected by agarose gel electrophoresis.
Poddar
Saibal K
SK
Pediatric Pharmacology Research Unit (PPRU), University of California at San Diego, La Jolla, CA 92093-0808, USA. spoddar@ucsd.edu
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D009980:Influenza A virus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D009981:Influenza B virus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D009691:Nucleic Acid Denaturation; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral; D012367:RNA, Viral