Lu H, Yi Tang, Lin Lin and David R Wolfgang
Surveillance for the Influenza A virus is the most important method used to monitor poultry and other animal species for the presence of the Influenza A viruses. Waterfowl and swine swab samples that tested positive for the Influenza A virus by real-time RT-PCR (rRT-PCR) but tested negative for the virus by virus isolation were further investigated in our recent studies using next-generation sequencing (NGS). A total of seven such pooled swab samples (four swine oral-pharyngeal (OPH) swab pools and three wild duck OPH and cloacal swab pools) were tested for influenza A virus by whole genome sequencing using NGS with the Illumina MiSeq system. Our sequencing results confirmed that none of these rRT-PCR positive samples (Ct-values of 22 to 28) contained Influenza A virus contigs; instead, all the samples contained multiple non-target contig sequences mismatched to the rRT-PCR primer and probe sequences, which led to the positive rRT-PCR results. These genome sequence findings provide scientific evidence that the rRT-PCR false positive results were caused by non-target contigs and not by target viral RNA. Additionally, these samples tested negative for the influenza A virus by conventional RT-PCR (cRT-PCR), which suggests cRT-PCR can serve as an alternative approach for identifying rRT-PCR false positive results.