Design, validation and absolute sensitivity of a novel test for the molecular detection of avian pneumovirus

This study describes attempts to increase and measure sensitivity of molecular tests to detect avian pneumovirus (APV). Polymerase chain reaction (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, and thereby 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 full-length DNA copies of APV genome derived from the same A-type virus. Four new nested PCR tests were designed in the fusion (F) protein [2 tests], small hydrophobic (SH) protein [1 test] and nucleocapsid (N) protein [1 test] genes and compared to an established test in the attachment (G) protein gene. Known amounts of full-length avian pneumovirus 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 the established G test, which proved able to detect 6000 copies of the G gene. The G test contained predominantly pyrimidine residues at its 3’ termini and because of this, oligonucleotides 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 the F gene.