Effect of preservative on recoverable RT-PCR amplicon length from influenza A virus in bird feces

Abstract

Surveillance for avian influenza A viruses in wild bird populations is often limited by requirements for a cold chain from time of specimen collection, by availability of ultra-low temperature specimen storage within a few hours or days, and by laborious classical virologic procedures. Successful storage of specimens in preservatives at ambient temperature and subsequent detection of RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) would assist in helping influenza surveillance efforts become more widespread in remote areas, as well as more timely and inexpensive. Here, we describe bird feces spiked with influenza A virus preserved with guanidine and commercial buffers or alcohols at ambient temperature and analyzed by RT-PCR protocols. Virus-specific RT-PCR products of, at most, 206 bp were recovered for samples preserved with alcohols and up to 521 bp for samples preserved with guanidine or commercial buffers. These results suggest that this approach is feasible in the field and that preserved specimens might be better assayed molecularly when preserved in guanidine or commercial buffers.

Fig. 1

Effect of preservative on RT-PCR amplification of various influenza viral amplicons in bird feces. Agarose gels stained with ethidium bromide show the RT-PCR amplification efficiencies for different amplicon sizes of influenza A/Puerto Rico/8/34 (H1N1) stored with various preservatives. The data shown are consistent with those results that were reproducible in five independent experiments. − = negative control (water template); + = positive control (viral RNA template); I = isopropanol preservative; E = ethanol preservative; M = methanol preservative; C = commercial preservative; G = guanidine buffer preservative. The left column gives amplicon size (bp). For the 137 bp product, upper band is specific, whereas the products underneath are probably primer dimers.