Optimierung und Evaluierung eines Loop-mediated Isothermal Amplification Assays zum Nachweis des Lebensmittelallergens Sellerie

Text in englischer SpracheZusammenfassung in deutscher SpracheLebensmittelallergien sind weitverbreitet und stellen für die Betroffenen ein großes Gesundheitsrisiko dar. Aus diesem Grund hat die Europäische Union Gesetze zur Kennzeichnungspflicht allergener Inhaltsstoffe erlassen, die sowohl verpackte als auch unverpackte Lebensmittel betreffen. Es sind bereits zahlreiche auf Antikörper bzw. auf DNA basierende Assays zum Nachweis der wichtigsten Lebensmittelallergene erhältlich, aber um auf eine schnelle und vor Ort durchführbare Methode zurückgreifen zu können, wurde ein bereits entwickelter Loop-mediated Isothermal Amplification (LAMP) Assay zum Nachweis von Apium graveolens in Lebensmitteln und prozessierten Produkten optimiert und evaluiert. Dieser Assay stellte sich als hochspezifisch für Sellerie heraus - mit einem Detektionslimit von 7,8 mg Selleriepulver pro Kilogramm gespiketer Lebensmittelprobe. Die Analyse von zehn kommerziell erhältlichen Lebensmittelprodukten und die Bestimmung einer Falsch-Negativ-Rate für extrahierte DNA aus drei verschiedenen Lebensmittelmatrizes, die mit DNA aus Sellerie gespiket wurde, erlaubten einen Vergleich des LAMP-Assays mit dem ausgewählten Referenz-PCR-Assay. Die LAMP-Reaktion wurde auch auf Heizblöcken durchgeführt, um den verlässlichen Nachweis des Analyten unabhängig vom verwendeten Gerät zu demonstrieren. Weiters wurden die Amplifikationsprodukte mittels Agarose-Gelelektrophorese und Fluoreszenzfärbung visualisiert, um die Ergebnisse mit jenen zu vergleichen, die durch das Echtzeit-Fluoreszenz-Monitoring produziert wurden. Diese Resultate zeigen, dass der LAMP-Assay zum Nachweis von Sellerie hochspezifisch sowie einfach und vor Ort durchführbar ist und dass er in Kombination mit einer einfachen Visualisierungsmethode das Potential hat, zu einem vielversprechenden Analyseinstrument für kommerzielle Anwendungen zu werden.Food allergies are widely distributed and pose a major health risk to affected people, which is why the European Union introduced labelling directives for packaged and unpackaged foods with regard to allergenic ingredients. There are already many immuno-based and DNA-based assays for the detection of the most important food allergens available, but in order to provide a rapid and onsite applicable method, an existing loop-mediated isothermal amplification (LAMP) assay for the detection of Apium graveolens in foods and products thereof was optimized and evaluated. The assay was shown to be highly specific for celery, and the limit of detection (LOD) was found to be as low as 7.8 mg celery powder per kilogram spiked food sample. The analysis of ten commercially available food products and a determination of the false-negative rate for the DNA extracted from three food matrices spiked with celery DNA allowed a comparison of the LAMP assay to the selected reference real-time PCR assay. The LAMP reaction was also performed on different heating blocks to demonstrate a reliable detection of the analyte independent from the instrument used. Furthermore, the amplification products were visualized via agarose gel electrophoresis and fluorescence staining in order to compare the results to those obtained from real-time fluorescence monitoring. These outcomes show that the LAMP assay for the detection of celery is highly specific, easy-to-perform and on-site applicable, and in combination with a simple visualization technique, it has the potential to become a promising tool for commercial uses.6

Challenges and perspectives in the application of isothermal DNA amplification methods for food and water analysis

Niederösterreichische Forschungs- und Bildungsgesellschaft (NFB), Life Science Call 2013Austrian Science Fund (FWF

Detection of a microbial source tracking marker by isothermal helicase-dependent amplification and a nucleic acid lateral-flow strip test

Abstract Over the last decades, various PCR-based methods have been proposed that can identify sources of faecal pollution in environmental waters. These microbial source tracking (MST) methods are powerful tools to manage water quality and support public health risk assessment. However, their application is limited by the lack of specialized equipment and trained personnel in laboratories performing microbiological water quality assessment. Here, we describe a novel molecular method that combines helicase-dependent amplification (HDA) with a strip test for detecting ruminant faecal pollution sources. Unlike quantitative PCR (qPCR), the developed HDA-strip assay only requires a heating block to amplify the ruminant-associated Bacteroidetes 16S rRNA marker (BacR). Following HDA, the reaction mixture can be directly applied onto the test strip, which detects and displays the amplification products by marker-specific hybridization probes via an on-strip colorimetric reaction. The entire assay takes two hours and demands no extensive practical training. Furthermore, the BacR HDA-strip assay achieved comparable results in head-to-head performance tests with the qPCR reference, in which we investigated source-sensitivity and source-specificity, the analytical limit of detection, and the sample limit of detection. Although this approach only yields qualitative results, it can pave a way for future simple-to-use MST screening tools

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Enterococcus spp. in water

Faecal pollution of water and the resulting potential presence of human enteric pathogens is a predominant threat to public health. Microbiological water quality can be assessed by the detection of standard faecal indicator bacteria (SFIB) such as E. coli or certain Enterococcus species. In recent years, isothermal amplification methods have become a useful alternative to polymerase chain reaction (PCR), allowing molecular diagnostics with simple or no instrumentation. In this study, a novel screening method for the molecular detection of Enterococcus spp. by loop-mediated isothermal amplification (LAMP) is described. A set of six specific LAMP primers was designed to amplify a diagnostic fragment of the Enterococcus 23S rRNA gene, which is present in several enterococcal species targeted by quantitative PCR (qPCR), which is the standard technique recommended by the US Environmental Protection Agency. Sensitivity and specificity tests were performed using a set of 30 Enterococcus and non-target bacterial reference strains. It is shown that LAMP is equally sensitive and even more specific than the qPCR assay. A dilution series of Enterococcus faecalis DNA revealed that the LAMP method can reliably detect 130 DNA target copies per reaction within 45 min. Additionally, enterococci isolated from Austrian surface waterbodies, as well as a set of DNA extracts from environmental waters, were tested. Contingency analysis demonstrated a highly significant correlation between the results of the developed LAMP assay and the reference qPCR method. Furthermore, a simple staining procedure with a fluorescence dye demonstrated the identification of amplified products by eye. In conclusion, this method is an important component for the efficient screening and testing of water samples in low-resource settings lacking sophisticated laboratory equipment and highly trained personnel, requiring only a simple heating block