10,458 research outputs found
Development of Diagnostic Tools for the Seed Potato Industry
No full textThe Australian potato industry is threatened by inadequate measures to control the virus health of seed potatoes. Potatoes are vegetatively propagated; therefore infection can result in disease spreading through generations. This has the potential to cause significant economic losses. Virus testing on tuber sprouts is currently conducted by ELISA, however a significant time delay of several weeks can occur while tubers sprout. There is a considerable need for a rapid, quantitative and cost effective virus test directly on bulked samples of dormant tubers to identify infected lots during seed multiplication.
The potato viruses of economic importance in Western Australia are Potato virus S, (PVS), Potato virus X, (PVX), Potato leafroll virus, (PLRV) and Tomato spotted wilt virus (TSWV). The main aim of this project was to develop reliable PCR-based methods for multiplex real-time quantitative detection of these viruses in bulked potato tuber samples for seed certification for domestic and export markets.
Knowledge of the distribution of the viruses within tuber tissue was needed to develop more effective methods of RNA extraction. The distribution of the viruses in histological sections of potato tubers was investigated using immunohistochemistry and in situ hybridization. All four viruses were found to be distributed at the stolon end of freshly harvested infected potato tubers. Extraction of RNA from tuber tissue is problematic because it contains starches and phenolics which inhibit RT-PCR. Extracting RNA from the tuber peelings would overcome this problem; however one of the viruses, PLRV, is restricted to the phloem in potato tubers. The distribution of the phloem in the superficial tissue of potato tubers was therefore investigated using histological staining and transmission electron microscopy. The vascular tissue was found to be within 2 mm below the epidermis of the tuber. With this knowledge, total RNA was extracted rapidly and efficiently from bulked potato peelings equivalent to 300 dormant tubers to detect single infections of PLRV, PVX, PVS and TSWV.
For the quantitative detection of these viruses in potato leaves and tuber tissue, specific primers and fluorescent-labeled TaqMan® probes were designed. A realtime multiplex, single tube RT-PCR assay was developed. The main tasks involved primer design, optimization of reagents, standardization of RNA samples from which standard curves for analysis were generated, and identification of a baseline on which to interpret results.
Limits of detection sensitivity were established using a range of virus transcript copy numbers (8 x 101 to 8 x 109 copies of PVX and PVS, 1 x 102 to 1 x 1010 copies of PLRV and 1 x 103 to 1 x 1010 copies of TSWV). The multiplexed assay was validated in blind studies. This high-throughput test is accurate and sensitive, and as a result this test is in the process of being commercialized and used by the seed potato industry of Western Australia as a cost-effective diagnostic tool to detect viruses reliably in bulked samples of dormant potato tubers
Paul Jones: A Feast Day for a Man of Peace
No full textA video covering Paul Jones, his history in Utah, his community work, pacifism, work as fourth Bishop of Utah, views on the First World War, his resignation, civil rights support, and continued work after resignatio
Murine oligonucleotide sequences for q-RT-PCR.
No full textMurine oligonucleotide sequences for q-RT-PCR.</p
RT-qPCR and qPCR assay details and performance for the viral targets.
No full textRT-qPCR and qPCR assay details and performance for the viral targets.</p
Prescribing by mental health nurses: the UK perspective
No full textPURPOSE. This article aims to discuss the growth of mental health nurse (MHN) prescribing in the United Kingdom as an exemplar for readers to compare progress in their own countries and context. This study also aims to provide a historical overview of this process in the United Kingdom where MHNs prescribe safely and competently.
CONCLUSIONS. Finally, evidence has shown that MHNs with prescriptive authority are competent when prescribing when compared to psychiatrists.
PRACTICE IMPLICATIONS. Despite organizational barriers and educational concerns, MHN prescribing is becoming embedded in the healthcare context in the United Kingdo
Belongingness: a prerequisite for nursing students’ clinical learning
No full textThe concept of belongingness has intuitive appeal. Human beings are social creatures; the need to belong and be accepted is fundamental, and social exclusion can be devastating. This paper reports on the selected findings from the qualitative phase of mixed-methods study that explored nursing students’ experience of belongingness while on clinical placements. The 18 interview participants in this study were from Australia and the United Kingdom. They provided a range of perspectives on belongingness and how it influenced their placement experience. Central to this discussion was their strong belief that belonging is a prerequisite for clinical learning. This theme dominated all of the interviews. Given that the primary purpose of clinical placements is for students to learn to nurse, there needs to be a clear understanding of the relationship between belongingness and learning. With reference to the published literature and excerpts from interview transcripts, this paper proposes that reconceptualising nursing students’ clinical learning experiences through a ‘lens of belongingness’ provides a new perspective and reveals yet unexplored insight
RT-LAMP primer investigation.
No full textSARS-CoV-2 RNA primer sets targeting the nucleopcapsid (N), envelope gene (E) (NEB design) and Orf1a (Rabe and Cepko Harvard Medical School) were tested independently and in combination (dual N+E reaction and multiplex Orf1a+N+E reaction) against 1x104 copies of Twist synthetic SARS-CoV-2 control RNA (T1 and T2). Negative control RNA from Betacoronavirus 1 strain OC43 and Influenza A (H1N1) at a single concentration (1x105 copies per well) plus a water no template control (NTC). A total RNA control primer set (NEB) targeting human beta actin (ACTB) was also included and tested against 5 ng total human RNA (hRNA). Both RT-LAMP fluorescent end-point and colorimetric 25 μl reactions were performed at 65°C for 40 minutes on a StepOnePlus thermoclycler. (A) Representative fluorescent amplification curves where time to positive (TTP) is the time at which amplification exceeds the manually set, reaction consistent threshold (red dotted line). (B) Representative colorimetric reactions whereby yellow indicates positive amplification and pink no amplification.</p
RT-PCR confirms indel mutations and identifies aberrant splice variants caused by CRISPR/Cas9 mutation.
No full text(A-D) WT and mutant allele RT-PCR products resolved on 2% agarose gel are shown in the left panels and splicing aberrations are depicted in the right panels. (A) The observed RT-PCR product for fancl_hg58 mutant was smaller in size (red arrow) than expected based on the 25 bp insertion in exon 8. The sequence of the hg58 product showed shift in its splice acceptor site resulting in loss of 48 bp sequence, that includes 23 bp from exon 8 beginning and CRISPR/Cas9 induced 25 bp insert. (B-D) Partial activation of cryptic splice site near the indel mutation was observed in fanca_hg41, fancb_hg42 and fancd1_hg45 mutant alleles. The RT-PCR products for these mutants showed two bands, one matched expected size (yellow arrows) and the other was from an altered splice product (red arrows). PCR products were cloned and sequenced to determine aberrant splice product sequence. In hg41 mutant, the indel mutation (2 bp del) in exon 12 results in partial skipping of mutated exon (B). In hg42 mutant, the indel mutation (4 bp del) in exon 1 results in partial use of new splice donor site in intron 1 (C). In hg45 mutant, the indel mutation (5 bp ins) in exon 10 results in partial use of new splice acceptor and donor in exon 10 (D). (E) The consequence on the predicted encoded protein based on the genomic indel mutation, and the observed aberrant splice product for all four mutant lines are shown. RT-PCR gel image data for all 36 alleles is shown in S3A Fig.</p
- …
