USE OF REVERSE GENETICS TO DEVELOP A POSITIVE CONTROL VIRUS FOR RT-NESTED PCR DETECTION OF SUBTYPE A AND B AVIAN METAPNEUMOVIRUS (AMPV)

A standardized RT-nested PCR of high sensitivity has been in use in our laboratory for some time to detect avian metapneumovirus subtype A and B. Until now we have avoided the use of positive control viruses (CV) because of the risk of contamination leading to sample false positives. The paper describes the production and testing of a modified virus which in our standard PCR produces RT-nested PCR amplicons of increased sizes compared to those generated from unmodified viruses, thus enabling cross contamination to the sample tested to be instantly detectable. A DNA copy of an AMPV subtype A genome was modified by site directed mutagenesis to introduce the subtype B G gene primer sequence (G9+B) at the equivalent position in the subtype A G gene. To increase amplicon sizes, an insertion was introduced between binding sites for the opposing primer pairs (outer pair G1+ and G6-, inner pairs G8+A and G5- or G9+B and G5-). PCR from the modified DNA produced nested amplicons of 463 and 556 bp compared to 268 for unmodified subtype A and 361 for unmodified subtype B viruses. Using reverse genetics the modified DNA generated a virus which, after RNA extraction, gave the same 463 and 556 bp amplicons in RT-nested PCRs. For further convenience virus was absorbed onto filter paper, dried and inactivated by microwave treatment, then stored in flip top tubes. RNA extracted from papers were henceforth used as reliable, efficient and distinguishable RT-nested PCR positive controls.