Detection of influenza A and B in respiratory secretions with the polymerase chain reaction

Abstract

Influenza A and B are RNA-containing viruses that frequently infect humans. Currently, sensitive detection of these viruses requires fresh respiratory secretions and special facilities for culture. To facilitate diagnosis of influenza, the polymerase chain reaction (PCR) was used in the present studies to detect DNA produced by reverse transcription of influenzal RNA in vaccines, tissue culture fluids, and stored respiratory secretions. Primers were directed at targets on the highly conserved segment 7 (matrix gene) of influenza A (212-bp product) and B (365-bp product). The primers were completely type specific. Critical variables in the assay were the concentration of pleotropic salts used during preparation of samples, the use of carrier RNA and RNase inhibitors during sample preparation, and the use of optimum K+ and Mg2+ levels at each step. Studies of 33 patients with symptoms of viral respiratory infection whose nasal washes had been cultured and frozen for up to 1 year before assay showed that PCR provided type-specific detection of influenza with a sensitivity comparable to that of culture of the fresh secretions. The assay offers a powerful test for detection of devitalized influenza viruses and may be useful in both diagnostic work and epidemiological studies of influenza.