NEW TEST FOR THE MOLECULAR DETECTION OF AVIAN PNEUMOVIRUS

This study describes attempts to measure and increase sensitivity of molecular tests which would be later used to detect Avian pneumovirus (APV) in field material. PCR diagnostic tests were designed for the detection of nucleic acid from an A type avian pneumovirus genome. The objective was selection of PCR oligonucleotide combinations which would provide the greatest test sensitivity, to enable optimal detection when used for later testing of field materials. Relative and absolute test sensitivities could be determined because of laboratory access to known quantities of purified DNA copies of the same A type full length APV genome. Four new nested PCR tests were designed in the fusion protein [2 tests] (F), small hydrophobic protein (SH) and nucleocapsid (N) protein genes and compared to an established test in the attachment protein (G) gene. Known amounts of full length APV genome were serially diluted (10 fold) and these dilutions were used as templates for the different tests. Sensitivities were found to differ between the tests, with the most sensitive being given by the established G test, which proved able to detect 8000 copies of G gene. The established G test contained predominantly pyrimidine residues at their 3’ termini and because of this, oligos for the most sensitive F test were modified to incorporate the same residue types at their 3’ termini. This was found to increase sensitivity so that after full 3’ pyrimidine substitutions, the F test became able to detect 600 copies of F gene. An RT-nested PCR test was then used to test limited field material. In this an RT step preceeded PCR testing which was identical to that used for DNA above. The modified F test again proved more sensitive than the established G test.