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초록·키워드
Type-specific rapid diagnosis of influenza is very important to proper treatments, prevention of epidemics, and world-wide epidemiological surveys. We evaluated the reverse transcription-polymerase chain reaction(RT-PCR) method for rapid detection of influenza virus, in comparison with the method of virus isolation and immunocapture enzyme-linked immunosorbent assay (ELISA). We used influenza A/Taiwan/1/86(H1N1), A/Beijing/39/92(H3N2), A/Shangdong/9/93 (H3N2), and B/Panama/45/90 strains as experimental viruses. Using isolation technique, we could detect live A/Beijing/32/92 virus 100,000 times more sensitively than HA test after 7 days of inoculation. Using immunocapture ELISA method, we were able to detect the virus 20 times more sensitively than HA test, and the type-specificities of immunocapture ELISA to type A and B were both 100%. In the RT-PCR method, we selected the primers which are complementary to type-specific region of the matrix protein coding viral cDNAs. After making the cDNA from influenza vRNA, 2×25 cycles of PCR reactions were performed with nested primers. The RT-PCR method was proved to be highly sensitive in detection of influenza virus, less than 10⁻⁴ HA unit/50μl concentration of influenza virus was detectable within 1 day, and it was 10,000 times more sensitive than that of HA test. There was no cross PCR detection between A and B viruses. We confirmed each virus band sequence by cycle sequencing and non-cloned T7 polymerase sequencing, reference to GenBank stored strains(A/Fort Monmouth/1/47 and B/Singapore/227/79). The phylogenetic study of type A virus revealed that the matrix protein genome sequence of 1994 Seoul epidemic strain(A/Seoul/94) was very similar to that of A/Shangdong/9/93 strain. These results suggest that RT-PCR provides a useful method of rapid detection and molecular epidemiologic study of human influenza viruses.
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UCI(KEPA) : I410-ECN-0101-2010-475-003015554
