Improved specificity for Giardia lamblia cyst quantification in wastewater by development of a real-time PCR method

International audienceThe protozoan parasite Giardia lamblia is the most common cause of waterborne disease outbreaks associated with drinking water in the United States. The conventional method used for the enumeration of Giardia cysts in water is based on immunofluorescence with monoclonal antibodies. It is tedious and time-consuming and has the major drawback to be non-specific for the only species infecting humans, G. lamblia. We have developed a real-time polymerase chain reaction (PCR) method using fluorescent TaqMan technology, which improved the specificity of G. lamblia cyst quantification compared to the immunofluorescence assay (IFA). However, this PCR was not totally specific for G. lamblia species and amplified Giardia ardeae target as well. This method showed a sensitivity of 0.45 cysts per reaction and an efficiency of 95% in purified suspensions. We have then applied this quantification method to raw wastewater, a medium containing numerous debris, particles and PCR inhibitors. The adaptation to these environmental samples was realized by a screening of three cyst purification methods and six DNA extraction protocols. Real-time quantification was accomplished by the simultaneous amplification of unknown samples and a tenfold serial dilution of purified G. lamblia cysts. For all samples, the concentrations observed with TaqMan PCR method were compared to the IFA values. Giardia spp. cysts were detected in all non-spiked raw wastewater samples with IFA procedure and the concentrations of Giardia spp. cysts used for the comparison between the two methods ranged between 3.3x10(2)/l and 4.3x10(3)/l. The highest TaqMan PCR/IFA ratios were observed when Percoll/sucrose flotation was combined with DNA extraction protocol optimized for cyst wall lysis, impurities adsorption on a resin, and double step protein digestion and column purification. The concentrations observed with this TaqMan PCR method ranged from 2.5x10(2) to 2.4x10(3) G. lamblia cysts/l and only one sample resulted in a no amplification curve. Thus, we developed a TaqMan PCR method increasing the rapidity and specificity of G. lamblia cyst quantification. The combination of Percoll/sucrose flotation and DNA extraction optimized protocol before TaqMan assay has provided a good indication of the G. lamblia contamination level in raw sewage samples

Legacy of contaminant N sources to the NO3− signature in rivers: a combined isotopic (δ15N-NO3−, δ18O-NO3−, δ11B) and microbiological investigation

International audienceNitrate content of surface waters results from complex mixing of multiple sources, whose signatures can be modified through N reactions occurring within the different compartments of the whole catchment. Despite this complexity, the determination of nitrate origin is the first and crucial step for water resource preservation. Here, for the first time, we combined at the catchment scale stable isotopic tracers (δ15N and δ18O of nitrate and δ11B) and fecal indicators to trace nitrate sources and pathways to the stream. We tested this approach on two rivers in an agricultural region of SW France. Boron isotopic ratios evidenced inflow from anthropogenic waters, microbiological markers revealed organic contaminations from both human and animal wastes. Nitrate δ15N and δ18O traced inputs from the surface leaching during high flow events and from the subsurface drainage in base flow regime. They also showed that denitrification occurred within the soils before reaching the rivers. Furthermore, this study highlighted the determinant role of the soil compartment in nitrate formation and recycling with important spatial heterogeneity and temporal variability

Fiabilisation de l’analyse de

La quantification de Legionella pneumophila dans les échantillons d’eaux résiduaires selon les méthodes normalisées existantes (normes NF T90-431 et XP T90-471, respectivement basées sur la mise en culture et la PCR quantitative) sont rendues difficiles, du fait notamment de l’hétérogénéité et la richesse en inhibiteurs (microorganismes susceptibles de générer une flore interférente et composés organominéraux inhibant l’amplification PCR). Or, la recherche des sources de contamination lors d’épisodes épidémiques de légionellose nécessite de disposer de méthodes fiables pour l’analyse de ce type de matrices. Sur la base de la méthode normalisée NF T90-431 (2003), les temps de contact et l ’ intensité des paramètres physico - chimiques (température, pH acide) instaurant une pression sélective favorable à la croissance des légionelles sur milieu de culture ont été optimisés. De même, l’application de la PCR quantitative sur ces matrices a nécessité l’évaluation et la comparaison de différents procédés d’extraction - purification d’ADN. Applicable en amont de la PCR, la séparation immunomagnétique (IMS), basée sur la reconnaissance spécifique antigène-anticorps, offre une solution alternative pour la concentration de L. pneumophila en permettant l’élimination d’une grande partie de la matière organique en suspension potentiellement inhibitrice de la PCR. Les développements analytiques ainsi obtenus permettront d’accéder à des matrices naturelles dont la complexité limitait le nombre d’analyses réalisées et empêchait jusqu’à présent de disposer de résultats fiables

Soil microbial diversity : an ISO standard for soil DNA extraction

International audienceSoil carries out functions that are crucial for the environment and life on earth and is therefore an essential non-renewable resource for mankind. Recently, the European Soil Framework Directive proposal (COM(2006)232) indicated that soil is under increasing environmental pressure mostly due to the intensification of human activities, which are damaging the capacity of soil to continue to perform in full its broad variety of crucial functions. Most of these soil functions are dependent on microorganisms inhabiting the soil. The diversity of soil microorganisms is the highest on earth with estimates of several thousand to several million different genomes per gram of soil (Whitman et al. 1998; Torsvik et al. 2002; Zhang and Xu 2008). However, fundamental understanding of the diversity and ecology of microbial communities carrying out soil functions has been hampered by our inability to grow most microorganisms under laboratory conditions.Since the early eighties, direct DNA-based methods have been developed to circumvent the biases resulting from the low number of microorganisms that could be cultured, thus hampering our understanding of microbial diversity in soil (Torsvik 1980; Porteous et al. 1991; Tsai et al. 1991; Smalla et al. 1993; Zhou et al. 1996; He et al. 2005). These methods were based on direct extraction of the DNA from micro-organisms living in the soil, using various lysis treatments. Since then, numerous articles have been published describing either new or improved methods for soil DNA extraction and at least ten companies are commercializing “Soil DNA” extraction kits. The on-going success of these direct molecular methods for studying soil micro-organisms is reflected by the fact that more than 1,000 articles are now published yearly using some type of soil DNA extraction method. Unfortunately, the wide use of these methods has resulted in a huge number of laboratory or even user-specific protocols, which contain minor to major modifications of the existing methods or kits.However, the choice of DNA extraction method is far from being inconsequential as it affects the picture of the microbial diversity present in a soil sample (Frostegard et al. 1999; Krsek et al. 1999; Martin-Laurent et al. 2001; He et al. 2005). To tackle this crucial problem and ensure comparable data, a standard soil DNA extraction method should be used. This is becoming all the more important because studies of soil microbial diversity based on soil DNA extraction are generating an exponential amount of sequence data thanks to rapid advances in sequencing technologies.For this purpose, we proposed in 2006 the standardization of soil DNA extraction to the International Organization for Standardization (ISO), with support from the French Standards Association and the French Environment and Energy Management Agency. The need for an international standard for soil DNA extraction has been rapidly recognized by ISO members as a priority and formally agreed upon. The ISO 11063 Soil quality method to directly extract DNA from soil samples is now being prepared by the Technical Committee ISO/TC 190, Subcommittee SC4, Biological methods. The standard was developed based on the publication made by Martin-Laurent et al. (2001). In the first phase, the technical scope of the future standard has been defined by a working group, consisting of expert researchers from countries interested in the subject matter. It was decided that since DNA purification is dependent on soil type, only the actual DNA extraction step, which is likely to generate the main differences between methods, would be subject to standardization. After a successful French inter-laboratory assay, we were requested during the ISO/TC 190 meeting in Delft in November 2008 to set up an international ring test for a final validation of the method. This task involved ten different laboratories from seven countries. Six soils collected in different European Countries were chosen for this study. The final report of this ring test was included in the revised of the standard and sent to the ISO in January 2010.After a 5 months ballot, more than two thirds of the votes cast by the national standards bodies members were positive and the ISO standard for Soil DNA extraction has been approved. The normative document will now be disseminated within the scientific community. However, adoption of the ISO standard is voluntary and both information and international cooperation will be needed for the acceptance and success of ISO/CD 11063 standard. We hope that this standardization of a soil DNA extraction method will be an important first step towards the unification of soil microbiologists

Essais inter-laboratoire pour évaluer le standard ISO 11063 ‘Qualité du sol – méthode pour l’extraction directe des acides nucléiques du sol’

L’extraction directe des acides nucléiques de microorganismes du sol est une étape cruciale de l’analyse moléculaire des communautés microbiennes du sol. Cependant, l’utilisation de nombreux protocoles d’extraction, chacun présent des biais, rendent difficile la comparaison des jeux de données. Cette déficience est d’autant plus dommageable que les méthodes moléculaires présentent un débit élevé et, en conséquence, génèrent de nombreuses données. Avec de surpasser cette problématique, en 2006 l’INRA a proposé à l’ISO une méthode d’extraction directe de l’ADN du sol. Cette méthode a été évaluée par 13 laboratoires européens indépendants au travers d’un essai interlaboratoire Français et d’un essai international. La reproductibilité de la méthode d’extraction des acides nucléiques du sol a été évaluée en comparant les quantités d’ADN, l’abondance et la structure génétique de communautés microbiennes extraits de 12 sols différents. Basés sur les résultats des essais interlaboratoires, la méthode a été approuvée à l’unanimité par les 21 pays votant à l’ISO. Le standard ISO11063 décrivant cette méthode a été publié par l’ISO. Il est en cours de traduction pour être publié en Français par l’AFNOR. Les résultats des essais interlaboratoires seront présentés et les perspectives offertes par ce nouveau standard en terme d’évaluation post-homologation de produits phytosanitaires discutées.Petric I., Philippot L., Abbate, Abbate C., Bispo A., Chesnot T., Hallin S., Laval K., Lebeau T., Lemanceau P., Leyval C., Lindstrom K., Pandard P., Romero E., Sarr A., Schloter M, Simonet P., Smalla K., Wilke B.M., Martin-Laurent F. 2011. Inter-laboratory evaluation of the ISO standard 11063 "Soil quality - Method to directly extract DNA from soil samples". J. Microbiol. Meth. 84:454-460

Inter-laboratory evaluation of the ISO standard 11063 "Soil quality -- Method to directly extract DNA from soil samples"

International audienceExtracting DNA directly from micro-organisms living in soil is a crucial step for the molecular analysis of soil microbial communities. However, the use of a plethora of different soil DNA extraction protocols, each with its own bias, makes accurate data comparison difficult. To overcome this problem, a method for soil DNA extraction was proposed to the International Organization for Standardization (ISO) in 2006. This method was evaluated by 13 independent European laboratories actively participating in national and international ring tests. The reproducibility of the standardized method for molecular analyses was evaluated by comparing the amount of DNA extracted, as well as the abundance and genetic structure of the total bacterial community in the DNA extracted from 12 different soils by the 13 laboratories. High quality DNA was successfully extracted from all 12 soils, despite different physical and chemical characteristics and a range of origins from arable soils, through forests to industrial sites. Quantification of the 16S rRNA gene abundances by real time PCR and analysis of the total bacterial community structure by automated ribosomal intergenic spacer analysis (A-RISA) showed acceptable to good levels of reproducibility. Based on the results of both ring-tests, the method was unanimously approved by the ISO as an international standard method and the normative protocol will now be disseminated within the scientific community. Standardization of a soil DNA extraction method will improve data comparison, facilitating our understanding of soil microbial diversity and soil quality monitoring