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

Extracting 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.

Optimised immunofluorescence procedure for enumeration of Cryptosporidium parvum oocyst suspensions.

The aim of this study was to evaluate an optimised immunofluorescence assay in terms of the variability of sets of counts for Cryptosporidium parvum oocyst suspensions and data recovery and the reliability of the procedure. A coefficient of variation (CV) of 10% was determined to be the maximum value acceptable for count variability. It was found that the optimised IFA tested provided a high precision for the sets of enumerations for suspensions containing 800-20,000 oocysts/mL. The procedure was found to be robust and providing high recovery level (96.3%). In terms of counting precision, the technique described here approaches the performance of flow cytometry and surpasses other manual techniques with a CV of 10% for a concentration close to 800 oocysts/mL. The procedure described is particularly suitable for the production of seed doses and for other applications requiring the titration of oocyst suspensions with a high degree of precision and accuracy.