Design, Validation, and Absolute Sensitivity of a Novel Test for the Molecular Detection of Avian Pneumovirus

Abstract

Abstract. 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 APV 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 with an established test in the attachment (G) protein 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, the most sensitive being the established G test, which proved able to detect 6,000 copies of the G gene. The G test contained predominantly pyrimidine residues at its 3Ј termini, and because of this, oligonucleotides for the most sensitive F test were modified to incorporate the same residue types at their 3Ј termini. This was found to increase sensitivity, so that after full 3Ј pyrimidine substitutions, the F test became able to detect 600 copies of the F gene.

Comparative evaluation of conventional RT-PCR and real-time RT-PCR (RRT-PCR) for detection of avian metapneumovirus subtype A

“…This fact could be explained by the presence of a pyrimidine residue at their 3' end in primers AMPVspecific targeting the F gene. This parameter was suggested to increase the sensitivity in some PCR primers designed to detect an AMPV/A cloned F gene (CECCHINATO et al, 2004). The sensitivity of the Nand F-based RRT-PCR seemed to be lower than the recently reported G-based RRT-PCR for AMPV/A detection (10 -1.5 TCID 50 mL -1 ;GUIONIE et al, 2007).…”

“…Molecular methods, such as reverse transcriptase-polymerase chain reaction (RT-PCR), allow the development of rapid, sensitive and specific detection of AMPV (BÄYON-AUBOYER et al, 1999;D'ARCE et al, 2005;DANI et al, 1999;GUIONIE et al, 2007;JUHASZ & EASTON, 1994). Different conventional RT-PCR were already developed by using primers defined either for the detection of all subgroups (BÄYON-AUBOYER et al, 1999;CECCHINATO et al, 2004), or for the specific identification of each of subgroups A-D (BÄYON-AUBOYER et al, 1999). In a recent study, sets of primers targeting attachment (G) gene and small hydrophobic (SH) gene were designed to identify the four AMPV subgroups by real time RT-PCR (RRT-PCR), which also provides the quantification of mRNAs (GUIONIE et al, 2007).…”

“…Different target genes can apparently alter the sensibility and specificity of virus detection by conventional (CECCHINATO et al, 2004) and RRT-PCR assays. Primers and probes targeting NS1, NP-1, and VP1 genes of Human bocavirus (HBoV) showed similar sensitivity and specificity in RRT-PCR assays (CHOI et al, 2008).…”