Detection and identification of human influenza viruses by the polymerase chain reaction

Abstract

A series of oligonucleotide primers are described which hybridize to conserved regions of influenza virus cDNA and prime DNA synthesis in Taq polymerase catalyzed amplification reactions (PCR). Primers were designed to hybridize as nested pairs and, following a two-step amplification, produce uniquely sized DNA fragments diagnostic for viral type and subtype. Influenza A and B matrix-protein genes and the influenza C haemagglutinin gene were targets for the type-specific primers. Subtypespecific primers targeted conserved sequences within the three haemagglutinin or two neuraminidase subtypes of different human influenza isolates. The utility of this method was demonstrated using computer search methods and by accurately amplifying DNA from a variety of influenza A, B, and C strains. Type-specific primer sets showed a broad type specificity and amplified DNA from viral strains of unknown sequence. Restriction mapping and DNA sequencing showed that fragments amplified in this manner derived from the input template, confirming the accuracy of the method and demonstrating how PCR can be used to quickly derive sufficient sequence information for analysis of viral relatedness. Subtyping primers were able to distinguish accurately between the three haemagglutinin (H1, H2, H3) and two neuraminidase (N1, N2) alleles of human influenza A isolates. Again DNA was amplified from viruses of unknown sequence confirming that most of these primer sets may prove useful as broad range subtyping reagents. In order to simplify the work associated with analysis of many samples, we have also devised a rapid method for the isolation of viral RNA and synthesis of cDNA. Using this ‘mini-prep’ technique, it is possible to detect, amplify, and identify picogram quantities of influenza virus in a single day, confirming that PCR provides a useful alternative to existing methods of influenza detection.