Design, optimisation and preliminary validation of a human specific loop-mediated amplification assay for the rapid detection of human DNA at forensic crime scenes.

The identification of samples at a crime scene which require forensic DNA typing has been the focus of recent research interest. We propose a simple, but sensitive analysis system which can be deployed at a crime scene to identify crime scene stains as human or non-human. The proposed system uses the isothermal amplification of DNA in a rapid assay format, which returns results in as little as 30min from sampling. The assay system runs on the Genie II device, a proven in-field detection system which could be deployed at a crime scene. The results presented here demonstrate that the system was sufficiently specific and sensitive and was able to detect the presence of human blood, semen and saliva on mock forensic samples.

Comparison of calibration methods for the quantification of Basmati and non-Basmati rice using microsatellite analysis.

The accurate quantification of non-Basmati rice in Basmati rice is central to the successful prosecution of adulteration, where non-Basmati rice has been substituted for Basmati rice. The current method and three alternatives of constructing calibration curves for the measurement of non-Basmati rice in Basmati rice using microsatellite analysis were investigated. The methods compared involved power regression, linear regression (with and without log(10) transformation) and hyperbolic regression of the ratio of Basmati to non-Basmati peak areas. Assessments were made using error uncertainty, standard error at the agreed limit of adulteration, and 95% confidence intervals for five example data sets. The linear regression of the ratio of peak areas to the ratio of content proportions was found to give the most precise calibration and thus enhanced quantification of the level of adulteration of Basmati rice with non-Basmati rice.

Effect of heat and pressure processing on DNA fragmentation and implications for the detection of meat using a real-time polymerase chain reaction.

The design of real-time polymerase chain reaction (PCR) assays for the detection of meat in processed products has focused on using small amplicons, often to the detriment of specificity. However, the relationship between amplification rates and the amplicon size for processed meat products has yet to be determined. To investigate this relationship, real-time PCR assays were designed to give a series of amplicons of increasing size. These assays were then used to assess amplification rates, in relation to amplicon size, in processed meat matrices. Although the most sensitive assays were those that used the smallest amplicons, amplification was still observed using amplicons of 351 base pairs for highly processed samples. It was found, therefore, that although in general, amplicons should be as small as possible, larger amplicons give efficient amplification and that small amplicons should not be chosen if they compromise assay specificity.

The detection of horse and donkey using real-time PCR.

We have developed real-time PCR assays specific for horse and donkey, applicable to the detection of low levels of horse or donkey meat in commercial products. Primers, designed to the mitochondrial cytochrome b gene, were 3' mismatched to closely related and other commercial species. Amplification of non-target species DNA was prevented by truncation of primers at the 5' position, thereby conferring complete specificity. Both assays were highly sensitive and detected the presence of 1pg of donkey template DNA or 25pg of horse template DNA when assessed using dilutions of DNA in water. Model food samples, spiked with horse or donkey muscle and commercial products containing horse, were successfully tested for the presence of horse or donkey, demonstrating the applicability of the assays to food products.

Truncation of oligonucleotide primers confers specificity on real-time polymerase chain reaction assays for food authentication.

The advent of real-time polymerase chain reaction has revolutionized the field of molecular biology, but the design and optimization of these assays has been largely overlooked in the literature. This dearth of information is probably in response to the provision of assay design software and robust guidelines issued by the leading manufacturer. However, many applications require highly specific assays with no cross-amplification of non-target DNA and it has been found that the software and guidelines, whilst producing assays of great sensitivity, do not necessarily produce specific assays. Two complementary strategies were used to confer specificity on a real-time assay, first by placing the 3' end of the primers on a point of sequence heterogeneity and, second, by truncating the primers at the 5' end to lower the calculated melting temperature. Using these strategies in concert, a specific assay for a conserved region of the mitochondrial cytochrome b gene was developed that can be used for the unambiguous detection of the target species in a meat mixture. This approach can be used for any real-time polymerase chain reaction assay to increase assay selectivity and specificity.

Rapid detection of chicken and turkey in heated meat products using the polymerase chain reaction followed by amplicon visualisation with vistra green.

A rapid and highly specific assay suitable for the routine detection of turkey and chicken in processed meat products has been developed. Based on PCR amplification of species-specific amplicons with rapid visualisation using vistra green, the assay may be completed within 5 h of receipt of sample. DNA was isolated from meat samples by the use of Wizard DNA isolation technology and followed by DNA amplification in the polymerase chain reaction using species specific primers, chicken forward (CF), chicken reverse (CR), turkey forward (TF) and turkey reverse (TR): the production of an amplicon was detected after the end of the PCR in less than 5 min using vistra green and a fluorescence plate reader. The presence of fluorescence denoted the presence of the target species in the sample.

Food allergy--towards predictive testing for novel foods.

The risks associated with IgE-mediated food allergy highlight the need for methods to screen for potential food allergens. Clinical and immunological tests are available for the diagnosis of food allergy to known food allergens, but this does not extend to the evaluation, or prediction of allergenicity in novel foods. This category, includes foods produced using novel processes genetically modified (GM) foods, and foods that might be used as alternatives to traditional foods. Through the collation and analysis of the protein sequences of known allergens and their epitopes, it is possible to identify related groups which correlate with observed clinical cross-reactivities. 3-D modelling extends the use of sequence data and can be used to display eptiopes on the surface of a molecule. Experimental models support sequence analysis and 3-D modelling. Observed cross-reactivities can be examined by Western blots prepared from native 2-D gels of a whole food preparation (e.g. hazelnut, peanut), and common proteins identified. IgEs to novel proteins can be raised in Brown Norway rat (a high IgE responder strain) and the proteins tested in simulated digest to determine epitope stability. Using the CSL serum bank, epitope binding can be examined through the ability of an allergen to cross-link the high affinity IgE receptor and thereby release mediators using in vitro cell-based models. This range of methods, in combination with data mining, provides a variety of screening options for testing the potential of a novel food to be allergenic, which does not involve prior exposure to the consumer.

Identification of peanut and hazelnut allergens by native two-dimensional gel electrophoresis.

A procedure for the native two-dimensional electrophoresis of peanut and hazelnut proteins is described. Proteins were solubilised after acetone treatment using a combination of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and tetramethylene sulphone. These extracts were analysed by a combination of isoelectric focusing in the presence of lactose in immobilized pH gradients followed by charge shift electrophoresis. Immunoblot analysis, using sera from nut allergic patients, allowed the identification of a peanut and hazelnut allergen with identical isoelectric point and apparent molecular mass. These proteins were recovered from duplicate gels using a mixture of formic acid, acetonitrile (ACN) and isopropanol. The molecular masses for both proteins, determined by matrix assisted laser desorption/ionisation-mass spectrometry (MALDI-MS), were 4826 Da.

Modification of the ion exchange HPLC procedure for the detection of nitrate and nitrite in dairy products.

Existing ion exchange HPLC methodology for nitrate and nitrite analysis in cured meat products suffers from high analyte variability at low concentrations and also chromatographic interference by artifacts in some other foods, such as dairy products. An investigation into the sources of variability has shown that both the cyclohexyl solid phase extraction cartridge and the glass fibre filter used in the original method can introduce artifacts which interfere with the determination of the nitrate in foodstuffs. We have also found that the use of a graphitized solid phase extraction cartridge used in tandem with the cyclohexyl solid phase extraction cartridge removed the artifacts from the chromatograph of dairy products that co-eluted with nitrite and nitrate. Values for the nitrite and nitrate content of dairy products were obtained by the HPLC procedure using these two solid phase extraction cartridges and the values obtained were in close agreement with those obtained by cadmium column reduction and colorimetry.