Design and Sensitivity of PCR Tests for Avian Pneumovirus

The first study describes attempts to increase and measure sensitivity of molecular tests to detect avian pneumovirus (APV). 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. In the second study a new nested PCR in the SH gene was designed and tested. This test was able to detect and distinguish the A and B type Avian Pneumovirus and was compared to the established one designed in the G gene. The new SH test was more sensitive for both A and B type of Avian Pneumovirus than the G test.