Key result
Modifying oligonucleotides for the F gene PCR test to incorporate 3' pyrimidine residues increased sensitivity, enabling the detection of 600 copies of the avian pneumovirus genome.
Incorporating pyrimidine residues at the 3' termini of PCR oligonucleotides significantly increased the sensitivity of a novel test for avian pneumovirus detection.
May improve avian pneumovirus PCR detection in research; leaves open clinical validation in field samples.
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 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.
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Cecchinato et al. (2004) studied Avian pneumovirus (APV) detection. Modified nested PCR test in the fusion (F) protein gene with 3' pyrimidine substitutions vs. Established test in the attachment (G) protein gene was evaluated on Absolute test sensitivity (detection limit of viral genome copies). Modifying oligonucleotides for the F gene PCR test to incorporate 3' pyrimidine residues increased sensitivity, enabling the detection of 600 copies of the avian pneumovirus genome.
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