title: An rRT-PCR assay to detect the matrix gene of a broad range of avian paramyxovirus serotype-1 strains.
abstract: The current U.S. Department of Agriculture (USDA)-validated real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay designed to detect the matrix gene of avian paramyxovirus serotype-1 (APMV-1) is the primary screening assay used in the United States. It has previously been shown to be unable to consistently detect all members of class I APMV-1. Diagnostic testing relies on rRT-PCR to quickly detect APMV-1 in wild birds, backyard flocks, live bird markets, commercial poultry, and for export testing. Limitations of the current USDA assay have raised concerns about the potential for some strains of APMV-1 to remain undetected by the primary screening assay. Mismatches in the probe were shown to cause a loss in template binding efficiency, resulting in lack of detection by the assay. Here, we describe the development and analytical validation of a new rRT-PCR assay designed to target a highly conserved region of the matrix gene across a wide range of APMV-1 strains. Limit of detection testing revealed a 3 log10 decrease in sensitivity for one low-virulence strain when compared to the USDA validated assay. Conversely, the assay showed increased sensitivity for a class I isolate and two virulent strains of APMV-1 that were not detected by the USDA-validated assay. The new assay also demonstrated a high degree of specificity by the lack of detection of 43 non-APMV-1 viruses.
authors.affiliation: National Veterinary Services Laboratories, Animal and Plant Health Inspection Service, U.S. Department of Agriculture, Ames, IA 50010, USA.
authors.forename: Nichole L
authors.initials: NL
authors.lastname: Hines
authors.forename: Mary Lea
authors.initials: ML
authors.lastname: Killian
authors.forename: Janice C
authors.initials: JC
authors.lastname: Pedersen
authors.forename: Monica M
authors.initials: MM
authors.lastname: Reising
authors.forename: Nestor A
authors.initials: NA
authors.lastname: Mosos
authors.forename: Christian
authors.initials: C
authors.lastname: Mathieu-Benson
authors.forename: Cathy L
authors.initials: CL
authors.lastname: Miller
mesh_terms: D000818:Animals; D001715:Bird Diseases / Q000175:diagnosis / Q000821:virology*; D001717:Birds; D008969:Molecular Sequence Data; D009521:Newcastle Disease / Q000175:diagnosis / Q000821:virology*; D009522:Newcastle disease virus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary; D012680:Sensitivity and Specificity; D014763:Viral Matrix Proteins / Q000235:genetics*
chemical_list: D014763:Viral Matrix Proteins