title: Primer modification improves rapid and sensitive in vitro and field-deployable assays for detection of high plains virus variants.
abstract: A high consequence pathogen, High plains virus (HPV) causes considerable damage to wheat if the crop is infected during early stages of development. Methods for the early, accurate, and sensitive detection of HPV in plant tissues are needed for the management of disease outbreaks and reservoir hosts. In this study, the effectiveness of five methods-real-time SYBR green and TaqMan reverse transcription-quantitative PCR (RT-qPCR), endpoint RT-PCR, RT-helicase dependent amplification (RT-HDA) and the Razor Ex BioDetection System (Razor Ex)-for the broad-range detection of HPV variants was evaluated. Specific PCR primer sets and probes were designed to target the HPV nucleoprotein gene. Primer set HPV6F and HPV4R, which amplifies a product of 96 bp, was validated in silico against published sequences and in vitro against an inclusivity panel of infected plant samples and an exclusivity panel of near-neighbor viruses. The primers were modified by adding a customized 22 nucleotide long tail at the 5' terminus, raising the primers' melting temperature (Tm; ca. 10°C) to make them compatible with RT-HDA (required optimal Tm = 68°C), in which the use of primers lacking such tails gave no amplification. All of the methods allowed the detection of as little as 1 fg of either plasmid DNA carrying the target gene sequence or of infected plant samples. The described in vitro and in-field assays are accurate, rapid, sensitive, and useful for pathogen detection and disease diagnosis, microbial quantification, and certification and breeding programs, as well as for biosecurity and microbial forensics applications.
authors.affiliation: National Institute for Microbial Forensics and Food & Agricultural Biosecurity, Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, Oklahoma, USA.
authors.forename: M
authors.initials: M
authors.lastname: Arif
authors.forename: G S
authors.initials: GS
authors.lastname: Aguilar-Moreno
authors.forename: A
authors.initials: A
authors.lastname: Wayadande
authors.forename: J
authors.initials: J
authors.lastname: Fletcher
authors.forename: F M
authors.initials: FM
authors.lastname: Ochoa-Corona
mesh_terms: D017931:DNA Primers / Q000235:genetics*; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000821:virology*; D010942:Plant Viruses / Q000235:genetics / Q000302:isolation & purification*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014908:Triticum / Q000821:virology*; D014773:Virology / Q000379:methods*
chemical_list: D017931:DNA Primers; D012367:RNA, Viral