Development of a quantitative PCR method to explore the historical occurrence of a nuisance microalga under expansion

A number of marine and freshwater harmful algal bloom (HAB) species have colonized new areas and expanded their habitat range in recent years. Nevertheless it is notoriously difficult to establish when colonization first occurred, what the dispersal routes are, and to separate recent invasion from increases in existent but small populations. The freshwater raphidophyte Gonyostomum semen is a nuisance species that has expanded its habitat range and increased in abundance in northern Europe during the past decades. To evaluate to what extent sediments can be used for determining historic occurrence of G. semen, a quantitative real-time PCR method for detecting cysts of this algae was developed. This paper presents a qPCR protocol with a set of primers that are specific to Gonyostomum and with PCR conditions optimized for sediment samples from humic lakes, which are the common habitat of G. semen. With this sensitive method as few as 1.6 cysts per PCR reaction could be reliably quantified, corresponding to 320 cysts per g wet weight sediment. Cysts were present in sediments with ages ranging from years to decades and their persistence allows detection of historic populations up to at least 50 years old. With this qPCR assay it will be possible to trace the presence of G. semen in environments prior to the onset of algae-specific monitoring programs as well as for quantification in water column samples.     

Comparative structural and functional analysis of genes encoding pectin methylesterases in Phytophthora spp.

We have scanned the Phytophthora infestans, P. ramorum, and P. sojae genomes for the presence of putative pectin methylesterase genes and conducted a sequence analysis of all gene models found. We also searched for potential regulatory motifs in the promoter region of the proposed P. infestans models, and investigated the gene expression levels throughout the course of P. infestans infection on potato plants, using in planta and detached leaf assays. We found that genes located on contiguous chromosomal regions contain similar motifs in the promoter region, indicating the possibility of a shared regulatory mechanism. Results of our investigations also suggest that, during the pathogenicity process, the expression levels of some of the analyzed genes vary considerably when compared to basal expression observed in in vitro cultures of non-sporulating mycelium. These results were observed both in planta and in detached leaf assays.     

基于TaqMan探针多重实时荧光PCR的肠道特殊菌群绝对定量方法的建立

本研究旨在建立对肠道主要共生菌的快速定量方法。根据细菌16S rRNA基因的保守序列并参考相关研究,设计针对总肠道菌群的通用引物和探针,以及针对双歧杆菌属、肠球菌属和肠杆菌科的特异性引物和探针,采用引物设计工具(Primer-BLAST) 以及聚合酶链式反应(polymerase chain reaction, PCR)扩增以验证引物和探针的特异性。通过分子克隆技术,构建各目标菌属目的基因的重组质粒作为qPCR检测的标准模板,选择标准模板进行重复性实验,并计算组内和组间重复变异系数。用所建立的方法对不同年龄段的临床粪便标本进行3类细菌的检测,并初步分析这些菌群与增龄的关系。结果显示,引物和探针具有较高的特异性;各目的细菌标准曲线在一定范围内线性关系良好,总菌群或各目标菌属的线性范围分别为：总菌群2.9×10³～2.9×10¹²copies/μL、双歧杆菌3.1×10²～3.1×10⁹ copies/μL、肠球菌5.9×10²～5.9×10⁹ copies/μL、肠杆菌6.3×10²～6.3×10⁹ copies/μL,决定系数R²≥ 0.995;检测的最低拷贝数为总菌群7.5×10² copies/μL、双歧杆菌 46 copies/μL、肠球菌 37 copies/μL、肠杆菌 51 copies/μL;重复性实验变异系数在1.32%以下,具有良好的可重复性。对不同年龄段临床粪便标本检测的结果显示,肠球菌和肠杆菌含量随增龄呈增长趋势,且老年组含量显著高于青年组,但在高龄老年人中未发现肠球菌数量增加。双歧杆菌含量随增龄减少,且各组间差异显著,在高龄老年人中也未观察到双歧杆菌显著减少。结果提示,本研究建立了一种可供临床推广的菌群绝对定量方法,能够同时检测3类细菌和菌群总量,对于菌群定量研究具有实际意义。     

Using propidium monoazide to distinguish between viable and nonviable bacteria, MS2 and murine norovirus

Aims: The ability to distinguish between viable and/or infectious micro-organisms and inactivated cells is extremely important for correctly performing microbial risk assessments. In this study, we evaluated whether propidium monoazide (PMA)-qPCR could distinguish between viable and nonviable bacteria and viruses. Methods and Results: A PMA-qPCR combined assay was applied to viable and inactivated bacteria (Escherichia coli and Bacillus subtilis) and viruses (MS2 and murine norovirus [MNV]). PMA, a DNA-intercalating agent, in combination with PCR was better able to distinguish between viable and nonviable bacteria and viruses than conventional PCR. Conclusions: These results suggest that a combined PMA-qPCR assay can be used to measure the viability of bacterial cells and bacteriophage MS2, but not MNV. Significance and Impact of the Study: PMA-qPCR could potentially be used to measure the viability of some micro-organisms, including virus. However, a thorough evaluation should be performed prior to measuring the viability of micro-organisms by PMA-qPCR in a quantitative way.     

Modelling bacterial transmission in human allergen-specific IgE sensitization

Aims: The impact of bacterial transmission from mother to child on human allergy development is poorly understood. The aim of the present work was therefore to use a temporal collected dataset of 117 mothers and their children to model the potential effect of mother‐to‐child bacterial transmission on allergy (IgE) sensitization. Methods and Results: We have recently shown a negative IgE correlation to high Escherichia coli levels until the age of 1 year, with a shift to positive correlation to high Bacteroides fragilis levels at the age of 2. In the present work, we used the previous published data to model the persistence and interaction effects of E. coli and B. fragilis with respect to IgE sensitization. Temporal modelling was made by first defining a stochastic model for sensitization state based on Markov chains and regression tree analyses. Subsequent simulations were used to determine the impact of mother‐to‐infant bacterial transmission. The regression tree analyses showed that E. coli colonization within 4 days was negatively correlated to sensitization, while lack of E. coli colonization at day 4 combined with B. fragilis colonization after 4 months was positively correlated. With Markov chain analyses, we found that E. coli was highly persistent in infants until the age of 4 months, while the persistence of B. fragilis increased with age. Conclusions: Simulations showed that the mother’s bacterial composition correlated significantly to the child’s IgE sensitization state at the age of 2 years. High E. coli and low B. fragilis levels in the mother were negatively correlated, while low E. coli and high B. fragilis were positively correlated to IgE. Significance and Impact of the Study: Our results support that allergy could partly be communicable, being transferred from mother to infant through the gut microbiota.