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 (T m ; ca. 10°C) to make them compatible with RT-HDA (required optimal T m ؍ 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.
Tobacco mosaic virus (TMV), Hosta virus X (HVX), Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) are a few of the major viruses that infect ornamental and nursery plants. These viruses cause significant losses that impact growers and the ornamental industry. Often, a single ornamental plant is co-infected by more than one virus, which makes identification and discrimination of these viruses a difficult task, thus creating delays and limiting regulatory measures for effective quarantine. The aim of this study is to develop a sensitive, rapid, economic, and reliable multiplex Reverse Transcription PCR (RT-PCR) for simultaneous detection and discrimination of these five viruses. Specific PCR primers were designed using the consensus sequences of corresponding capsid protein (CP) genes of HVX and CMV, the nucleocapsid protein (NP) genes of TSWV and INSV, and the movement protein (MP) and CP genes of TMV. The primers were validated in vitro using single and multiplex RT-PCR assays. The detection limit of each primer set in multiplex RT-PCR was 100 fg (TMV), 1 fg (HVX), 10 fg (CMV), 10 pg (TSWV) and 10 pg (INSV). Forty-six infected nursery samples collected from different locations in the USA were screened for virus infections using this multiplex RT-PCR. The multiplex RT-PCR has a potential for its application in routine diagnostics, quarantine, and epidemiological studies. The developed method is reliable, sensitive, and economic for testing a wide range of ornamental and nursery plants for detection of these viruses.
Nanoparticles of magnetite passivated with gelatin and starch were synthesised using a co-precipitation technique. The nanoparticles were characterised using ultraviolet-visible (UV-vis), dynamic light scattering (DLS), Zeta potential, transmission electron microscope (TEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The UVvis spectra showed characteristic surface plasmon resonance of magnetite nanoparticles. The DLS results showed the nanoparticles to have average hydrodynamic diameters of 138 ± 2 and 283 ± 21 nm for particles passivated with gelatin and starch, respectively. The stability in a colloidal solution was greater in nanoparticles passivated with gelatin than nanoparticles obtained with starch, as can be seen by their Zeta potential value (−31 ± 2 and −16 ± 0.5 mV, respectively). According to the TEM evaluation, the use of gelatin allowed to obtain nanoparticles with a spherical morphology and an average size of 10 ± 2 nm. However, when using starch the nanoparticles exhibited diverse morphologies with an average size of 25 ± 7 nm. The XRD results confirmed the crystalline structure of the samples, which showed crystallite sizes of 14.90 and 24.43 nm for nanoparticles passivated with gelatin and starch, respectively. FTIR analysis proved the establishment of interactions between functional groups of biopolymers and magnetite nanoparticles.
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