Development and validation of qualitative SYBR®Green Real-Time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes

A combination of four qualitative SYBR®Green qPCR screening assays targeting two levels of discrimination: Listeria genus (except Listeria grayi) and Listeria monocytogenes, is presented. These assays have been developed to be run simultaneously using the same polymerase chain reaction (PCR) programme. The paper also proposes a new validation procedure to specifically validate qPCR assays applied to food microbiology according to two guidelines: the ISO 22118 norm and the “Definition of minimum performance requirements for analytical methods of GMO testing”. The developed assays target the iap, prs and hlyA genes that belong to or neighbour the virulence cluster of Listeria spp. The selected primers were designed to amplify short fragments (60 to 103 bp) in order to obtain optimal PCR efficiency (between 97 and 107 % efficiency). The limit of detection of the SYBR®Green qPCR assays is two to five copies of target genes per qPCR reaction. These assays are highly accurate (98.08 and 100 % accuracy for the Listeria spp. and L. monocytogenes assays, respectively).     

Development of a duplex real-time PCR for differentiation between E. coli and Shigella spp

Aims: The aim of this study was to develop a real‐time PCR test for differentiation between Shigella spp. and E. coli, in particular enteroinvasive Escherichia coli (EIEC). Methods and Results: A duplex real‐time PCR specific for the genes encoding for β‐glucuronidase (uidA) and lactose permease (lacY) was developed. Ninety‐six isolates including 11 EIEC isolates of different serotypes and at least three representatives of each Shigella species were used for selectivity testing. All isolates tested were positive for the uidA gene. Additionally, all E. coli isolates were positive for the lacY gene, whereas no Shigella isolate tested harboured lacY. Conclusions: The duplex real‐time PCR assay was found to be simple, rapid, reliable and specific. Significance and Impact of the Study: If possible at all, delineation of so‐called inactive EIEC from Shigella spp. is cumbersome. Biochemical and serological methods are limited to specific pheno‐ and serotypes. This assay clearly simplifies the differentiation of both.     

Response of a shallow boreal lake to recent nutrient enrichment – implications for diatom-based phosphorus reconstructions

Water quality of the shallow, mesotrophic, and macrophyte-dominated Lake Kaljasjärvi has been monitored at three to four year intervals since 1978. During the monitoring period, surface-water total phosphorus (TP) concentrations have typically varied between 20 and 25 g P l^–1. However, elevated total phosphorus concentrations were measured in 1987, 1991, and 1999. Diatom-based reconstruction of the historical lake-water TP concentrations was therefore employed to study the recent development of the lake. However, the diatom-TP model did not predict the high measured phosphorus concentrations despite the changes observable in diatom assemblages. In addition, the ratio of sedimentary diatom remains to chrysophycean stomatocysts declined towards the top of the sediment core, indicating decreasing trophy rather than eutrophication. Analysis of sedimentary pigments and phosphorus fractions, used to examine further the changes, also produced results that contradicted the simple eutrophication hypothesis. In particular, the proportion of chlorophyll derivatives instead of carotenoids increased and there was a rise in the concentration of refractory instead of NaOH-extracted phosphorus. These features appear to be related to the extensive littoral areas of the lake since enhanced littoral production can explain both the observed changes in sediment chemistry and the low diatom-inferred TP (DI-TP). Littoral primary producers are suggested to have benefited from the increased phosphorus inputs to the lake, transferring some of the phosphorus to the detrital pool and contributing to the increased pigment concentrations of sedimentary organic matter. High proportions of non-planktonic diatoms in the samples lower DI-TP because periphytic taxa are assigned low TP optima in the inference models used. Abundant aquatic macrophytes may also have made the lake resistant to eutrophication by assimilating nutrients, providing refuge for zooplankton, and having an allelopathic effect on phytoplankton. Since 1980, however, the sedimentary diatom assemblages also indicate increasingly eutrophic conditions. Additional loading from numerous cottages during the last 20 years seems to have caused observable changes in the phytoplankton communities.     

Response to “On the choice of methodology for evaluating dose-rate effects on radiation-related cancer risks” by Walsh et al.

Radiation and Environmental Biophysics -     

Easy and rapid method of zygosity determination in transgenic mice by SYBR<Superscript>®</Superscript> Green real-time quantitative PCR with a simple data analysis

Establishment and maintenance of transgenic mouse strains require being able to distinguish homozygous from heterozygous animals. To date, the developed real-time quantitative PCR techniques are often complicated, time-consuming and expensive. Here, we propose a very easy and rapid method with a simple data analysis to determine zygosity in transgenic mice. We show that the real-time quantitative PCR using SYBR Green fluorescent dye can be applied to discriminate two-fold differences in copy numbers of the transgene. Our procedure has to fit only three simple requirements: (1) to design primers capable of detecting one Ct difference for two-fold differences in DNA amounts (2) to measure genomic DNA concentrations accurately and (3) to have a reference animal of known zygosity in each run. Then, if the Ct values for the control gene are similar in all samples, we are able to compare directly the Ct values for the transgene in every sample, and so, to deduce the zygosity status of each mouse relative to the reference animal. This method is really simple and reliable, and it may be valuable as a rapid screening tool for zygosity status in transgenic animals.     

Design of a core–shell type immuno-magnetic separation system and multiplex PCR for rapid detection of pathogens from food samples

We report an immuno-magnetic separation system developed by the immobilization of pathogen-specific antibodies on the core–shell magnetic beads. The magnetic beads were grafted with glycidylmethacrylate (GMA) using surface-initiated atom transfer radical polymerization (SI-ATRP). For immuno-magnetic separation (IMS) of target bacterial cells from others, antibodies for Escherichia coli and Salmonella enterica serovar Typhimurium cells were immobilized on the magnetic beads via glutaraldehyde coupling reaction. Our IMS system successfully separated Salmonella cells when the concentrations of target (i.e., Salmonella) and interfering (i.e., E. coli) cells were at the same level. Polymerase chain reaction (PCR) assays amplifying the rfb/rfbE region of the E. coli genome and a 647-bp fragment of the invA region of Salmonella were performed as the specific selection to accurately confirm the presence of E. coli and Salmonella, respectively. IMS and multiplex PCR methods can be used for specific and quantitative detection of pathogens from food samples. Thus, this study developed a reliable and direct system for rapid detection of Salmonella and E. coli in food samples. In addition, IMS method could be easily adapted to detect other pathogens by selecting the pertinent antibody.     

Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet “Beta vulgaris L.”: GMO application

Key message

Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis.

Abstract

The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R ² > 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.     

Optimization of medium composition for the production of alkaline β-mannanase by alkaliphilic Bacillus sp. N16-5 using response surface methodology

In this work, a 2² factorial design was employed combining with response surface methodology (RSM) to optimize the medium compositions for the production of alkaline β-mannanase by alkaliphilic Bacillus sp. N16-5 isolated previously from sediment of Wudunur Soda Lake in Inner Mongolia, China. The central composite design (CCD) used for the analysis of treatment combinations showed that a second-order polynomial regression model was in good agreement with experimental results, with R ² = 0.9829 (P < 0.05). The maximum activity was obtained at NaCl concentration (84.4 g l⁻¹) and sodium glutamate (3.11 g l⁻¹) and a high medium pH around 10.0. Under such conditions, the activity of alkaline β-mannanase achieved 310.1 U/ml in the scale of 5-l fermenter, which was increased nearly twice compared with the original. Through optimization, the substrates shifted from the expensive substrates, such as locust bean gum and peptone, to the inexpensive ones such as konjac powder, soymeal, and sodium glutamate. The experiment results also suggested that the environmental conditions of high salinity and high alkalinity, as well as the inducer substrates, play very important roles in the production of the alkaline β-mannanase by alkaliphilic Bacillus sp. N16-5.     

Medium optimization by response surface methodology for poly-γ-glutamic acid production using dairy manure as the basis of a solid substrate

Dairy manure, supplemented with agro-industrial materials, was used as the solid substrate for high yield of poly-γ-glutamic acid (γ-PGA) by Bacillus subtilis CCTCC202048. The solid-state fermentation medium was optimized by response surface methodology. In the first optimization step, a Plackett–Burman design was used to evaluate the influence of related factors. Wheat bran, soybean cake and glutamic acid were found to be more compatible supplement with dairy manure and positively influenced on γ-PGA production. In the second step, the concentrations of the three supplemental nutrients above were further optimized using a Box–Behnken design. The average γ-PGA yield (4.70%) in triplicate under optimal conditions was obtained on the laboratory scale, whereas it was 3.58% at compost experiment. These would lay a foundation for lessening the pollution of dairy manure, increasing fertilizer efficiency and exploring a late-model organic fertilizer that retains water and nutrients.     

An ‘on–off–on’ fluorescent switch based on a luminous covalent organic framework for the rapid and selective detection of glyphosate

Glyphosate, the most used herbicide in the world, has a residue problem that cannot be ignored. However, glyphosate itself does not have fluorescence emission and lacks the conditions for fluorescence detection. In this work, a rapid and selective fluorescence detection method of glyphosate was designed by an ‘on–off–on’ fluorescent switch based on a luminous covalent organic framework (L-COF). Only the fixed concentration of Fe³⁺ as an intermediate could trigger the fluorescent switch and no incubation step was required. The proposed method showed good accuracy with a correlation coefficient of 0.9978. The method's limits of detection and quantitation were 0.88 and 2.93 μmol/L, which were lower than the maximum allowable residue limits in some regulations. Environmental water samples and tomatoes were selected as actual samples to verify the application in a complex matrix. A satisfactory mean recovery from 87% to 106% was gained. Furthermore, Fe³⁺ could induce fluorescence quenching of L-COF through the photo-induced electron transfer (PET) effect, while the addition of glyphosate could block the PET effect to achieve detection. These results demonstrated the proposed method had abilities to detect glyphosate and broaden the application of L-COF.     

Reply and explanation to Little et al. “Response to: On the choice of methodology for evaluating dose‑rate effects on radiation‑related cancer risks”

Radiation and Environmental Biophysics -     

Expression patterns of WRKY genes in di-haploid Populus simonii × P. nigra in response to salinity stress revealed by quantitative real-time PCR and RNA sequencing

Key message

Spatio-temporal expression patterns of 13 out of 119 poplar WRKY genes indicated dynamic and tissue-specific roles of WRKY family proteins in salinity stress tolerance.

Abstract

To understand the expression patterns of poplar WRKY genes under salinity stress, 51 of the 119 WRKY genes were selected from di-haploid Populus simonii × P. nigra by quantitative real-time PCR (qRT-PCR). We used qRT-PCR to profile the expression of the top 13 genes under salinity stress across seven time points, and employed RNA-Seq platforms to cross-validate it. Results demonstrated that all the 13 WRKY genes were expressed in root, stem, and leaf tissues, but their expression levels and overall patterns varied notably in these tissues. Regarding overall gene expression in roots, the 13 genes were significantly highly expressed at all six time points after the treatment, reaching the plateau of expression at hour 9. In leaves, the 13 genes were similarly up-regulated from 3 to 12 h in response to NaCl treatment. In stems, however, expression levels of the 13 genes did not show significant changes after the NaCl treatment. Regarding individual gene expression across the time points and the three tissues, the 13 genes can be classified into three clusters: the lowly expressed Cluster 1 containing PthWRKY28, 45 and 105; intermediately expressed Clusters 2 including PthWRKY56, 88 and 116; and highly expressed Cluster 3 consisting of PthWRKY41, 44, 51, 61, 62, 75 and 106. In general, genes in Cluster 2 and 3 displayed a dynamic pattern of “induced amplification—recovering”, suggesting that these WRKY genes and corresponding pathways may play a critical role in mediating salt response and tolerance in a dynamic and tissue-specific manner.     

Response by H. H. Pattee to Jon Umerez’s Paper: “Where Does Pattee’s “How Does a Molecule Become a Message?” Belong in the History of Biosemiotics?”

Umerez’s analysis made me aware of the fundamental differences in the culture of physics and molecular biology and the culture of semiotics from which the new field of biosemiotics arose. These cultures also view histories differently. Considering the evolutionary span and the many hierarchical levels of organization that their models must cover, models at different levels will require different observables and different meanings for common words, like symbol, interpretation, and language. These models as well as their histories should be viewed as complementary rather than competitive. The relation of genetic language and human language is the central issue. They are separated by 4 billion years and require entirely different models. Nevertheless, these languages have in common a unique unlimited expressive power that allows open-ended evolution and creative thought. Understanding the nature of this expressive power and how it arises remains a basic unsolved problem of biosemiotics.