AimsTo perform an international trial to derive alert and action levels for the use of quantitative PCR (qPCR) in the monitoring of Legionella to determine the effectiveness of control measures against legionellae.Methods and ResultsLaboratories (7) participated from six countries. Legionellae were determined by culture and qPCR methods with comparable detection limits. Systems were monitored over ≥10 weeks. For cooling towers (232 samples), there was a significant difference between the log mean difference between qPCR (GU l−1) and culture (CFU l−1) for Legionella pneumophila (0·71) and for Legionella spp. (2·03). In hot and cold water (506 samples), the differences were less, 0·62 for Leg. pneumophila and 1·05 for Legionella spp. Results for individual systems depended on the nature of the system and its treatment. In cooling towers, Legionella spp. GU l−1 always exceeded CFU l−1, and usually Legionella spp. were detected by qPCR when absent by culture. The pattern of results by qPCR for Leg. pneumophila followed the culture trend. In hot and cold water, culture and qPCR gave similar results, particularly for Leg. pneumophila. There were some marked exceptions with temperatures ≥50°C, or in the presence of supplementary biocides. Action and alert levels for qPCR were derived that gave results comparable to the application of the European Guidelines based on culture. Algorithms are proposed for the use of qPCR for routine monitoring.ConclusionsAction and alert levels for qPCR can be adjusted to ensure public health is protected with the benefit that remedial actions can be validated earlier with only a small increase in the frequency of action being required.Significance and Impact of the StudyThis study confirms it is possible to derive guidelines on the use of qPCR for monitoring the control of legionellae with consequent improvement to response and public health protection.

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During maturation of mammalian brain, variants of both linker (i.e. H1°) and core (i.e. H3.3) histone proteins accumulate in nerve cells. As the concentration of the corresponding transcripts decreases, in postmitotic cells, even if the genes are actively transcribed, it is likely that regulation of variant histone expression has relevant post-transcriptional components and that cellular factors affect histone mRNA stability and/or translation. Here we report that PIPPin, a protein that is highly enriched in the rat brain and contains a coldshock domain, binds with high specificity to the transcripts that encode H1°and H3.3 histone variants. Both mRNAs are bound through the very end of their 3-untranslated region that encompasses the polyadenylation signal. Although PIPPin is present both in the cytoplasm and the nucleus of nerve cells, PIPPin-RNA complexes can be obtained only from nuclear extracts. The results of two-dimensional electrophoretic analysis suggest that a relevant proportion of nuclear PIPPin is more acidic than expected, thus suggesting that its RNA binding activity might be modulated by post-translational modifications, such as phosphorylation.During development of an organism and tissue differentiation, chromatin must be remodeled to permit entrance of transcription factors and hence expression of genes at the right places and times. Although a critical moment for setting new patterns of chromatin organization is the S phase of the cell cycle, it is now clear that chromatin can be remodeled also in the absence of DNA replication, by energy consuming complexes (1-4). The possibility that remodeling also allows entrance, at topologically defined regions of the nucleus, of specific histone isotypes, which might locally modify chromatin organization even more, is provocative and deserves of attention.We previously demonstrated that, in the developing rat brain, the concentration of H1°and H3.3 mRNAs decreases between the embryonal day 18 (E18) and the postnatal day 10 (P10), whereas the corresponding genes are transcribed at the same rate at any stage studied, suggesting that the two genes are regulated mainly at post-transcriptional level (5, 6). As post-transcriptional control processes, including regulation of splicing (7), vectorial transport of mature mRNAs (8 -10), regulation of mRNA stability (11-13), and availability for translation (14, 15), are mediated by several classes of RNA-binding proteins (for review, see , it is likely that developing rat brain contains mRNA-binding factors involved in the binding and regulation of mRNAs encoding histone variants. We reported in a previous paper (19) cloning and analysis of a cDNA encoding a putative RNA-binding protein, specifically expressed in the rat brain and conserved from Drosophila melanogaster to man. The protein, that contains two regions with chemical homology to double-stranded RNA-binding motifs (16) was called PIPPin after the first four amino acids of the second of these motifs (PIPP, in one-letter code).Here we report that PIPPin c...

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The RNA purification is the gold standard for the detection of SARS-CoV-2 in swab samples, but it is dependent on the availability of chemical reagents. In this study, we evaluated the heat treatment method without RNA extraction as a reliable option to nucleic acid purification.