Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan(®) assays and pyrosequencing, while quantitative, cannot detect less than ∼1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan(®) probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan(®) probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment.

The large-scale use of neonicotinoid insecticides has raised growing concerns about their potential adverse effects on farmland birds, and more generally on biodiversity. Imidacloprid, the first neonicotinoid commercialized, has been identified as posing a risk for seed-eating birds when it is used as seed treatment of some crops since the consumption of a few dressed seeds could cause mortality. But evidence of direct effects in the field is lacking. Here, we reviewed the 103 wildlife mortality incidents reported by the French SAGIR Network from 1995 to 2014, for which toxicological analyses detected imidacloprid residues. One hundred and one incidents totalling at least 734 dead animals were consistent with an agricultural use as seed treatment. Grey partridges (Perdix perdix) and “pigeons” (Columba palumbus, Columba livia and Columba oenas) were the main species found. More than 70% of incidents occurred during autumn cereal sowings. Furthermore, since there is no biomarker for diagnosing neonicotinoid poisonings, we developed a diagnostic approach to estimate the degree of certainty that these mortalities were due to imidacloprid poisoning. By this way, the probability that mortality was due to poisoning by imidacloprid-treated seeds was ranked as at least “likely” in 70% of incidents. As a result, this work provides clear evidence to risk managers that lethal effects due to the consumption by birds of imidacloprid-treated seeds regularly occur in the field. This in turn raises the question of the effectiveness of the two main factors (seed burying and imidacloprid-treated seeds avoidance) that are supposed to make the risk to birds negligible. Risk factors and the relevance of mitigation measures are discussed.Electronic supplementary materialThe online version of this article (doi:10.1007/s11356-016-8272-y) contains supplementary material, which is available to authorized users.

Estimating exposure of wild birds to plant protection products is of key importance in the risk assessment process evaluating their harmful potential. In this paper, we propose an ecologically-relevant methodology to estimate potential exposure to active substances (ASs) of a farmland focal bird, the gray partridge Perdix perdix. It is based on bird habitat use of fields at the time of pesticide applications. It accounts for spatio-temporal heterogeneity at population and landscape scales. We identify and quantify the potential exposure to 179 ASs of 140 clutches during pre-laying, laying, and incubation phases, and of 75 coveys. The data come from a large scale field study combining radiotelemetry and a farmer survey. They were collected in 12 different representative sites. The proportion of clutches potentially exposed to a given chemical was ≥5% for 32 ASs; prothioconazole and epoxiconazole ranking first. 71% of clutches were potentially exposed to ≥1 AS and 67% to ≥2 ASs. Mixtures involved 2 to 22 ASs. They emerged from commercial formulations, tank mixtures, bird habitat use, and combinations. ASs were fungicides (53%), herbicides (25%), and insecticides (16%) used on a variety of crops in April-June, when ground-nesting birds are breeding. The European Food Safety Authority conclusions report a long-term first-tier toxicity-to-exposure ratio (TERlt) <5 for 11 out of 19 documented ASs, and higher-tier TERlt <5 for 5 out of 10 ASs. This suggests a potential risk for bird reproduction in farmlands. Globally 13% of coveys were potentially exposed to 18 ASs during the first month (1-4 coveys per AS). The use of our field data in future research and risk assessment is discussed.