The Potential of Synthetic Biology for Trichoderma reesei.

Within the last 20 years, ground-breaking progress has been made in the field of synthetic biology, enabling the construction of novel pathways up to entire synthetic genomes in both prokaryotic and eukaryotic organisms. These innovations are primarily adapted for biotechnological applications, where filamentous fungi such as Trichoderma reesei are widely used to produce various enzymes of industrial interest. In the following chapter, we provide a broad overview on the current progress involving this particular organism, covering studies on synthetic promoters and transcription factors as well as synthetic expression platforms. Furthermore, this chapters aims to be a short introduction to the present book since many methods mentioned here are described in detail in the subsequent chapters.

Simple lysis of bacterial cells for DNA-based diagnostics using hydrophilic ionic liquids.

The extraction of nucleic acids from microorganisms for subsequent molecular diagnostic applications is still a tedious and time-consuming procedure. We developed a method for the rapid preparation of genomic DNA from bacteria based on hydrophilic ionic liquids (ILs). First, we tested eight ILs in different buffer systems for their inhibitory effects on quantitative PCR. The cell lysis potential of different IL/buffer combinations was assessed by application on Enterococcus faecalis as a model organism for Gram-positive bacteria. The two best ILs, choline hexanoate and 1-ethyl-3-methylimidazolium acetate, were compared with the reference enzymatic method and two commercial DNA extraction kits. All methods were evaluated on four Gram-positive and four Gram-negative bacterial species that are highly relevant for environmental, food, or clinical diagnostics. In comparison to the reference method, extraction yields of the IL-based procedure were within one order of magnitude for most of the strains. The final protocol for DNA extraction using the two ILs is very low-cost, avoids the use of hazardous chemicals and can be performed in five minutes on a simple heating block. This makes the method ideal for high sample throughput and offers the opportunity for DNA extraction from bacteria in resource-limited settings or even in the field.

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

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.

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

Molecular diagnostic tools in the field of food and water quality analysis are becoming increasingly widespread. Usually, based on DNA amplification techniques such as polymerase chain reaction (PCR), these methods are highly sensitive and versatile but require well-equipped laboratories and trained personnel. To reduce analysis time and avoid expensive equipment, isothermal DNA amplification methods for detecting various target organisms have been developed. However, to make molecular diagnostics suitable for low-resource settings and in-field applications, it is crucial to continuously adapt the working steps associated with DNA amplification, namely sample preparation, DNA extraction, and visualization of the results. Many novel approaches have been evaluated in recent years to tackle these challenges, e.g., the use of ionic liquids for the rapid isolation of nucleic acids from organisms relevant for food and water analysis or the integration of entire analytical workflows on microfluidic chips. In any event, the future of applications in the field of isothermal amplification will probably lie in ready-to-use cartridges combined with affordable handheld devices for on-site analysis. This trend article aims to make prospective users more familiar with this technology and its potential for moving molecular diagnostics from the laboratory to the field. Graphical abstract ᅟ.

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.

A Complementary Isothermal Amplification Method to the U.S. EPA Quantitative Polymerase Chain Reaction Approach for the Detection of Enterococci in Environmental Waters.

We report a novel molecular assay, based on helicase-dependent amplification (HDA), for the detection of enterococci as markers for fecal pollution in water. This isothermal assay targets the same Enterococcus 23S rRNA gene region as the existing quantitative polymerase chain reaction (qPCR) assays of U.S. Environmental Protection Agency Methods 1611 and 1609 but can be entirely performed on a simple heating block. The developed Enterococcus HDA assay successfully discriminated 15 enterococcal from 15 non-enterococcal reference strains and reliably detected 48 environmental isolates of enterococci. The limit of detection was 25 target copies per reaction, only 3 times higher than that of qPCR. The applicability of the assay was tested on 30 environmental water sample DNA extracts, simulating a gradient of fecal pollution. Despite the isothermal nature of the reaction, the HDA results were consistent with those of the qPCR reference. Given this performance, we conclude that the developed Enterococcus HDA assay has great potential as a qualitative molecular screening method for resource-limited settings when combined with compatible up- and downstream processes. This amplification strategy can pave the way for developing a new generation of rapid, low-cost, and field-deployable molecular diagnostic tools for water quality monitoring.

Detection of the food allergen celery via loop-mediated isothermal amplification technique.

Since 2005, celery and celery products have to be labeled according to Directive 2003/89/EC due to their allergenic potential. In order to provide a DNA-based, rapid and simple detection method suitable for high-throughput analysis, a loop-mediated isothermal amplification (LAMP) assay for the detection of celery (Apium graveolens) was developed. The assay was tested for specificity for celery since closely related species also hold food relevance. The limit of detection (LOD) for spiked food samples was found to be as low as 7.8 mg of dry celery powder per kilogram. An evaluation of different amplification and detection platforms was performed to show reliable detection independent from the instrument used for amplification (thermal cycler or heating block) and detection mechanisms (real-time fluorescence detection, agarose gel electrophoresis or nucleic acid staining). The analysis of 10 commercial food samples representing diverse and complex food matrices, and a false-negative rate of 0% for approximately 24 target copies or 0.08 ng celery DNA for three selected food matrices show that LAMP has the potential to be used as an alternative strategy for the detection of allergenic celery. The performance of the developed LAMP assay turned out to be equal or superior to the best available PCR assay for the detection of celery in food products.

Detection of the 35S promoter in transgenic maize via various isothermal amplification techniques: a practical approach.

In 2003 the European Commission introduced a 0.9% threshold for food and feed products containing genetically modified organism (GMO)-derived components. For commodities containing GMO contents higher than this threshold, labelling is mandatory. To provide a DNA-based rapid and simple detection method suitable for high-throughput screening of GMOs, several isothermal amplification approaches for the 35S promoter were tested: strand displacement amplification, nicking-enzyme amplification reaction, rolling circle amplification, loop-mediated isothermal amplification (LAMP) and helicase-dependent amplification (HDA). The assays developed were tested for specificity in order to distinguish between samples containing genetically modified (GM) maize and non-GM maize. For those assays capable of this discrimination, tests were performed to determine the lower limit of detection. A false-negative rate was determined to rule out whether GMO-positive samples were incorrectly classified as GMO-negative. A robustness test was performed to show reliable detection independent from the instrument used for amplification. The analysis of three GM maize lines showed that only LAMP and HDA were able to differentiate between the GMOs MON810, NK603, and Bt11 and non-GM maize. Furthermore, with the HDA assay it was possible to realize a detection limit as low as 0.5%. A false-negative rate of only 5% for 1% GM maize for all three maize lines shows that HDA has the potential to be used as an alternative strategy for the detection of transgenic maize. All results obtained with the LAMP and HDA assays were compared with the results obtained with a previously reported real-time PCR assay for the 35S promoter in transgenic maize. This study presents two new screening assays for detection of the 35S promoter in transgenic maize by applying the isothermal amplification approaches HDA and LAMP.