5.1 Initial identification of influenza infection
Oral swabs or nasal aspirates are initially screened for the presence of a variety of respiratory viruses. This is done by extracting RNA from the sample and subjecting it to a reverse-transcription polymerase chain reaction (RT-PCR) as described below:
- Initially the RNA sample is reverse transcribed into complementary DNA (cDNA), using a commercially-available reverse transcriptase enzyme.
- The cDNA is then used in a standard PCR reaction to detect and amplify a short sequence of nucleotides specific to the virus. Multiple DNA sequences, each specific for a different type of virus, can be amplified in the same reaction, provided that these sequences are of different lengths.
- Each of the different amplified sequences is separated from the others when the entire sample is subjected to gel electrophoresis (an analytical technique in which molecules of different sizes move at various rates through a gel support in an applied electric field, thus making it possible to identify specific molecules.)
The polymerase chain reaction (PCR) technique is illustrated in Video 2.
Typically, nucleotide sequences specific to five types of virus are searched for in each sample: influenza A, influenza B, respiratory syncytial virus (Baltimore group V, (–)ssRNA virus, and a major cause of respiratory illness in young children), adenoviruses and enteroviruses. Those samples that test positive for influenza in the RT-PCR reaction are inoculated into cells in culture. Sufficient virus for a limited number of tests can be produced from such cultures within 24 hours, but they are often maintained for up to a week.