Fast and sensitive aflatoxin B1 and total aflatoxins ELISAs for analysis of peanuts, maize and feed ingredients

Aflatoxins are a group of carcinogenic compounds produced by Aspergillus fungi that can grow on different agricultural crops. Both acute and chronic exposure to these mycotoxins can cause serious illness. Due to the high occurrence of aflatoxins in crops worldwide fast and cost-effective analytical methods are required for the identification of contaminated agricultural commodities before they are processed into final products and placed on the market. In order to provide new tools for aflatoxin screening two prototype fast ELISA methods: one for the detection of aflatoxin B₁ and the other for total aflatoxins were developed. Seven monoclonal antibodies with unique high sensitivity and at the same time good cross-reactivity profiles were produced. The monoclonal antibodies were characterized and two antibodies showing IC₅₀ of 0.037 ng/mL and 0.031 ng/mL for aflatoxin B₁ were applied in simple and fast direct competitive ELISA tests. The methods were validated for peanut matrix as this crop is one of the most affected by aflatoxin contamination. The detection capabilities of aflatoxin B₁ and total aflatoxins ELISAs were 0.4 μg/kg and 0.3 μg/kg for aflatoxin B₁, respectively, which are one of the lowest reported values. Total aflatoxins ELISA was also validated for the detection of aflatoxins B₂, G₁ and G₂. The application of the developed tests was demonstrated by screening 32 peanut samples collected from the UK retailers. Total aflatoxins ELISA was further applied to analyse naturally contaminated maize porridge and distiller's dried grain with solubles samples and the results were correlated with these obtained by UHPLC-MS/MS method.

Establishing an EGFR mutation screening service for non-small cell lung cancer - sample quality criteria and candidate histological predictors.

INTRODUCTION: EGFR screening requires good quality tissue, sensitivity and turn-around time (TAT). We report our experience of routine screening, describing sample type, TAT, specimen quality (cellularity and DNA yield), histopathological description, mutation result and clinical outcome. METHODS: Non-small cell lung cancer (NSCLC) sections were screened for EGFR mutations (M+) in exons 18-21. Clinical, pathological and screening outcome data were collected for year 1 of testing. Screening outcome alone was collected for year 2. RESULTS: In year 1, 152 samples were tested, most (72%) were diagnostic. TAT was 4.9 days (95%confidence interval (CI)=4.5-5.5). EGFR-M+ prevalence was 11% and higher (20%) among never-smoking women with adenocarcinomas (ADCs), but 30% of mutations occurred in current/ex-smoking men. EGFR-M+ tumours were non-mucinous ADCs and 100% thyroid transcription factor (TTF1+). No mutations were detected in poorly differentiated NSCLC-not otherwise specified (NOS). There was a trend for improved overall survival (OS) among EGFR-M+ versus EGFR-M- patients (median OS=78 versus 17 months). In year 1, test failure rate was 19%, and associated with scant cellularity and low DNA concentrations. However 75% of samples with poor cellularity but representative of tumour were informative and mutation prevalence was 9%. In year 2, 755 samples were tested; mutation prevalence was 13% and test failure only 5.4%. Although samples with low DNA concentration (2.2 ng/μL), the mutation rate was 9.2%. CONCLUSION: Routine epidermal growth factor receptor (EGFR) screening using diagnostic samples is fast and feasible even on samples with poor cellularity and DNA content. Mutations tend to occur in better-differentiated non-mucinous TTF1+ ADCs. Whether these histological criteria may be useful to select patients for EGFR testing merits further investigation.

Enzyme-induced Aggregation of Plasmonic Nanoparticles for the Development of Label-free and Ultrasensitive Detection of Bacterial DNA

Sensitive detection of pathogens is critical to ensure the safety of food supplies and to prevent bacterial disease infection and outbreak at the first onset. While conventional techniques such as cell culture, ELISA, PCR, etc. have been used as the predominant detection workhorses, they are however limited by either time-consuming procedure, complicated sample pre-treatment, expensive analysis and operation, or inability to be implemented at point-of-care testing. Here, we present our recently developed assay exploiting enzyme-induced aggregation of plasmonic gold nanoparticles (AuNPs) for label-free and ultrasensitive detection of bacterial DNA. In the experiments, AuNPs are first functionalized with specific, single-stranded RNA probes so that they exhibit high stability in solution even under high electrolytic condition thus exhibiting red color. When bacterial DNA is present in a sample, a DNA-RNA heteroduplex will be formed and subsequently prone to the RNase H cleavage on the RNA probe, allowing the DNA to liberate and hybridize with another RNA strand. This continuously happens until all of the RNA strands are cleaved, leaving the nanoparticles ‘unprotected’. The addition of NaCl will cause the ‘unprotected’ nanoparticles to aggregate, initiating a colour change from red to blue. The reaction is performed in a multi-well plate format, and the distinct colour signal can be discriminated by naked eye or simple optical spectroscopy. As a result, bacterial DNA as low as pM could be unambiguously detected, suggesting that the enzyme-induced aggregation of AuNPs assay is very easy to perform and sensitive, it will significantly benefit to development of fast and ultrasensitive methods that can be used for disease detection and diagnosis.