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 HPV... More
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.
