title: Avian metapneumovirus RT-nested-PCR: a novel false positive reducing inactivated control virus with potential applications to other RNA viruses and real time methods.
abstract: Using reverse genetics, an avian metapneumovirus (AMPV) was modified for use as a positive control for validating all stages of a popular established RT-nested PCR, used in the detection of the two major AMPV subtypes (A and B). Resultant amplicons were of increased size and clearly distinguishable from those arising from unmodified virus, thus allowing false positive bands, due to control virus contamination of test samples, to be identified readily. Absorption of the control virus onto filter paper and subsequent microwave irradiation removed all infectivity while its function as an efficient RT-nested-PCR template was unaffected. Identical amplicons were produced after storage for one year. The modified virus is likely to have application as an internal standard as well as in real time methods. Additions to AMPV of RNA from other RNA viruses, including hazardous examples such HIV and influenza, are likely to yield similar safe RT-PCR controls.
authors: Department of Infection Biology, Faculty of Health and Life Sciences, University of Liverpool, Leahurst Campus, Neston, Cheshire, CH64 7TE, UK.
authors: Marco
authors: M
authors: Falchieri
authors: Paul A
authors: PA
authors: Brown
authors: Elena
authors: E
authors: Catelli
authors: Clive J
authors: CJ
authors: Naylor
mesh_terms: D000818:Animals; D005189:False Positive Reactions; D029121:Metapneumovirus / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods / Q000592:standards