EcoDiagnostics strives to be at the forefront of developing technology in order to provide our clients with the key strategic advantages that this can offer. Collectively our directors have over 40 years experience in the development and application of DNA-based technology in the diagnostic fields of food safety, environmental and veterinary testing. Some of the key advantages of utilising DNA based methods include a high level of test specificity and sensitivity and the ability to rapid delivery of results. Many of our existing clients are realising the commercial advantages of this suit of technologies for the detection of problematic biological organisms that may otherwise require lengthy culture times or may not be culturable at all on existing selective culture media. The fact that DNA-based diagnostic technology has become increasingly affordable in recent years, coupled to our investment in a highly automated and efficient laboratory set up means that we can now deliver extremely competitive pricing across a wide range of DNA-based testing solutions customised to our clients requirements.
Whilst recognising the value of DNA based technology EcoDiagnostics also understands the value and importance of more established technologies especially when meeting statutory testing standards . As such we also offer expertise in a range of technologies including the microbiological culture of a wide range of yeast, mould and bacterial organisms in addition to ELISA testing for a range of problematic substances in a wide variety of matrices.
Our state-of-the art NATA-accredited laboratory is fully equipped with the latest in automated robotic technology for both DNA extraction and amplication using techniques such as PCR and real-time PCR, DNA barcoding and DNA meta-barcoding. Our laboratory facilities also include a fully contained microbiological culture suite and ELISA testing laboratory. Our highly trained laboratory personnel have over 50 years combined experience in the laboratory testing sector.
MICROBIOLOGICAL CULTURE PLATING
Microbiological culture involves the spreading of a biological sample onto the surface a pre-prepared culture plate (often made of agar) so that microbiological organism (eg bacteria, yeast or fungi) selectively grow and can thus be detected, identified and potentially quantified. Microbes need different nutrients to grow including water, a source of energy, sources of carbon, sulphur, nitrogen, phosphorus, certain minerals, and other vitamins and growth factors. The components of the growth media on a particular agar plate as well as the incubation temperature determine which microbes will grow. Once viable microbial organisms have grown on a plate they can be counted, and their identify verified if necessary using biochemical or DNA-based tests.
Enzyme-linked immunosorbent assay (ELISA) is based on the detection of specific proteins in a sample (eg an allergen such as gluten in a food substance) by a specific capture antibody (Fig 1; 1), Capture antibodies are linked to each well of a 96-well plate and the sample added to each well such that if present an antibody-antigen-antibody (sandwich) complex is formed (Fig 1; 2). An enzyme is then linked to this complex followed by a chromogen substrate for the enzyme (Fig 1; 3). Bound enzyme conjugate converts the colourless chromogen into a blue product (Fig 1; 4). The addition of the stop reagent leads to a color change from blue to yellow. The measurement is made photometrically at 450 nm. The absorbance is proportional to the protein concentration of the sample.
POLYMERASE CHAIN REACTION
The polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Amplification of millions of copies allows the specific and sensitive detection of very few copies of a gene present in the original sample. Following the prior extraction of DNA from a sample, the method involves multiple heating and cooling cycles to 1) denature double stranded DNA; 2) anneal highly specific short priming sequences to these single strands and 3) extension of the priming sequences using a copying enzyme to regenerate double the previous number of double stranded DNA molecules. Amplified products are traditionally stained with a dye that binds to DNA and separated by size using electrophoresis and agarose gels.
REAL-TIME POLYMERASE CHAIN REACTION
Real-time PCR (or qPCR) is based on the principles of traditional PCR but differs in that is amplification of DNA is detected in real-time as it happen. Key advantages include:
Highly specific- Methods involve the use of a highly specific probe in addition to specific primers that binds its complementary target in the reaction mix. As PCR amplification proceeds and strand copying occurs this probe is cleaved off which results in the generation of light that is detected by lasers on the qPCR instrument.
– Highly sensitive- reaction chemistry and use of laser detection is such that methods are highly sensitive and can detect a single copy of DNA from an organism in a sample.
– Quantitative- detecting amplification in real-time allows comparison against standards during the exponential phase of the reaction (rather than at the end) when the amount of amplification (r light generated) is directly proportional to the DNA copy number in the starting material
– Rapid- The detection of amplification in real-time removes the requirement for detecting products using electrophoresis meaning that the reaction can be completed in as little as 30mins.
DNA barcoding is a taxonomic method that uses a short genetic marker in an organism’s DNA to identify it as belonging to a particular species. It is based on the amplification of that specific gene region using PCR from an individual biological organism (including plants, animals and microbes), followed by the determination of its DNA sequence. This DNA sequence is compared to databases that hold information of taxonomically verified specimens that have had these corresponding and unique genetic sequences characterised. Advantages include:
– Ability to identify an organism where taxonomic Identification is not possible (eg when homogenised in food materials, only part of an organism is available, sample is degraded or decomposed, or when no taxonomic features are not evident – eg eggs or juvenile stages)
– Cost effective method to identify difficult organisms where specialist taxonomic identification may not be readily accessible (eg for biosecurity threats)
– Highly accurate
DNA metabarcoding is based on the same principle as DNA barcoding. However, instead of individual specimen identification, the technique is used to simultaneously characterise all organism in a mixed biological sample at the same time, making it highly efficient. Generic primer sets are used that are capable of amplifying the DNA barcodes of a selected group of organisms (eg bacteria, marine pests, meiofauna etc) in a biological sample (eg a food or drink sample, a biosecurity trap sample, a water sample). PCR amplification is conducted using these primers and all of the generated products are sequenced using the latest Next Generation Sequencing methods. This generates literally millions of individual barcode sequences that are then analysed to provide a comprehensive picture on the biological composition of the sample. Key advantages include:
– Cost effective profiling of biological samples (eg to identify problematic organisms in a system or process)
– Ability to investigate the effect of different treatments on biological communities (eg disinfection or remediation regimes)