Molecular detection of Phytophthora pluvialis, the causal agent of red needle cast in Pinus radiata.

BACKGROUND: Phytophthora pluvialis was first described in 2013 and is the causal agent of red needle cast (RNC) in Pinus radiata as well as infection in Douglas fir (Pseudotsuga menziesii). A species-specific PCR is necessary for detection of this pathogen and diagnosis of RNC. OBJECTIVE: To design and validate a species-specific molecular assay for P. pluvialis using isolates from infected pine needles. METHODS: Species-specific PCR primers were generated from the ras-related GTP-binding protein 1 gene (ypt1) gene sequence, concentrating on DNA regions unique to P. pluvialis, and real-time and quantitative polymerase chain reaction (qPCR) were used to detect P. pluvialis from both artificially inoculated and naturally infected samples. RESULTS: The species-specific PCR assay was generated following P. pluvialis DNA sequence analysis. In vitro tests of the specificity of the probe-based, quantitative, polymerase chain reaction (qPCR) assay showed that no amplification was observed with other Phytophthora species including other closely-related clade 3 species, or with fungal species associated with pine or with pine DNA. The limit of detection of the qPCR assay was 2 pg/µl. When the qPCR assay was used to detect P. pluvialis in artificially-inoculated and naturally infected P. radiata needles, a PCR product was detected in all inoculated samples; the mean concentration ranges of P. pluvialis DNA in the inoculated and naturally infected samples tested were 5.9-124.5 pg/µl and 8.1-340.2 pg/µl, respectively. The assays described herein were used with serological diagnostic strips, providing the ability to identify to species level. CONCLUSIONS: The assay described herein detects P. pluvialis with high specificity and sensitivity from a range of DNA samples, including those extracted from infected plant material and serological diagnostic strips. The ability to detect and identify P. pluvialis, from infected tissues directly, provides value and practicality to diagnostics, biosecurity and research.

Are Irish General Practitioners minimising infection risk from sharps?

A self administered postal questionnaire was sent to a computer randomised sample of 200 GPs registered with the Irish College of General Practitioners. The objective of this was to determine if Irish GPs are minimising infection risk from sharps, by observing current phlebotomy techniques, current sharps disposal methods and by availing of Hepatitis B vaccination. The response rate was 69% (n = 138). Phlebotomy technique: syringes and needles were used by 55% and the vacutainer system by 34% with 11% using both methods. Of note, 50% of G.P.s usually recapped needles and 57% never wore gloves. Only 48% of G.P.s had been vaccinated against Hepatitis B. More recently qualified G.P.s were more likely to be vaccinated. Many Irish G.P.s have unsafe phlebotomy practices. There is inadequate use of specifically designed impenetrable sharps boxes, with no standard method of either collection or disposal of used sharps. Uptake of Hepatitis B vaccination is low. Amongst Irish G.P.s, there is a gap between the perceived and the actual safety of sharps disposal methods in use. No protocol advising G.P.s on infection control exists. We recommend that guidelines regarding safe phlebotomy technique, sharps disposal and the importance of Hepatitis B vaccination be produced and strongly reinforced, and that within each health board there is a standard policy and procedure established for collection and disposal of used sharps. The above are essential for the health and safety for both G.P.s and the public.