'Phytochip': on-chip detection of phytopathogenic RNA viruses by a new surface plasmon resonance platform.

The surface plasmon resonance (SPR) based 'Phytochip' was developed to distinguish virus-infected plants from non-infected plants. The system detects DNA-RNA hybridization to show the presence of phytopathogenic viruses such as the RNA virus barley stripe mosaic virus (BSMV) in wheat leaves. To achieve this BSMV and wheat specific oligonucleotides, and a negative control yeast oligonucleotide, were immobilized on a SPR gold surface chip. After optimization of the hybridization parameters with purified wheat samples, wheat infected with BSMV resulted in detectable signals with both the BSMV and the wheat probes. In contrast, a hybridization reaction was not be detected with the negative probe. The method is fast and sensitive with a detection time of 3000s (50min), a detection limit of 14.7pgµl(-1) BSMV RNA and a measuring range of 14.7-84pgµl(-1) BSMV RNA (1.323-7.56ng BSMV RNA per 90µl sample). These characteristics, combined with the high throughput design, make it suitable for application in plant breeding and virus control.

Evaluation and validation of a novel Arxula adeninivorans estrogen screen (nAES) assay and its application in analysis of wastewater, seawater, brackish water and urine.

A novel Arxula adeninivorans yeast estrogen screen (nAES) assay has been developed for detection of estrogenic activity in various liquid samples such as wastewater, seawater, brackish water and swine urine. Two bio-components were engineered to co-express the human estrogen receptor α (hERα) and an inducible reporter gene; either the non-conventional phytase gene (phyK, derived from Klebsiella sp. ASR1) or the non-conventional tannase gene (ATAN1, derived from Arxula). Both reporters were put under the control of an Arxula derived glucoamylase (GAA) promoter, which was modified by the insertion of two estrogen-responsive elements (EREs). The Arxula transformation/expression platform Xplor® 2, which lacks resistance markers and E. coli elements, was used to select stable mitotic transformants. They were then analyzed for robustness and suitability as the bio-component for the nAES assay. Two types of the nAES assay based on the reporter proteins phytase and tannase (nAES-P, nAES-T) were used in this work. The nAES-P type is more suitable for the analysis of seawater, brackish water and urine whereas the nAES-T type exhibited higher robustness to NaCl. Both assay types have similar characteristics for the determination of estrogen in sewage and urine samples e.g. 6-25 h assay period with detection and determination limits and EC(50) values for 17ß-estradiol of 2.8 ng L(-1), 5.9 ng L(-1), 33.2 ng L(-1) (nAES-P) and 3.1 ng L(-1), 6.7 ng L(-1) and 39.4 ng L(-1) (nAES-T). Substrate specificity and analytical measurement range (AMR) for both assay types are also similar. These characteristics show that the nAES assay based on non-conventional salt tolerant yeast is applicable for a high throughput estrogen analysis in the environmental and regulatory control sectors.