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/Bejing/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 10E-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 corss PCR detection between A and
Bviruses.
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.
|