Species-specific detection

The following sections provide information on how these techniques have been employed to set up species-specific detection systems for potentially OTA-producing fungi. 

Schilling, A. G., Moeller, E. M., and Geiger, H. H. (1996). Polymerase chain reaction-based assays for species-specific detection of Fusarium culmorum, F. graminearum and f. avena-ceum. Phytopathology 86, 515-522. 

Fig. 3. Comparison of different enzyme-linked immuno sorbent assay (ELISA) methods adapted for immuno-polymerase chain reaction (IPCR). Dependent on the purification grade of the sample to be analyzed and the availability of specific and functionalized antibodies, several typical ELISA protocols were adapted to IPCR. In the direct approach (A), the pure antigen is immobilized to the microplate surface and subsequently detected by a labeled specific antibody. If no labeled antibody is available (e.g., because of unpurified ascites fluid containing the antibody or loss in activity following labeling), a standardized labeled secondary species-specific antibody is used for detection of the primary antigen-specific antibody (B). For the detection of the antigen from matrices such as serum, plasma, tissue homogenate, and so on, a capture antibody immobilized to the microplate surface was used either in a direct (C) or indirect (D) sandwich approach, with the latter one additionally including a secondary species-specific detection antibody. For different methods of coupling antibody and DNA, abbreviated by in this figure, compare Fig. 2. Note that protein A chimeras (Fig. 2A) are not compatible with capture antibodies (Fig. 3C, D).

Statistical process control methods are applied to preparative chromatography for the case where cut points for the effluent fractions are determined by on-line species-specific detection (e.g., analytical chromatography). A simple, practical method is developed to maximize the yield of a desired component while maintaining a required level of product purity in the presence of measurement error and external disturbances. Relations are developed for determining tuning parameters such as the regulatory system gain. 

Also shown in Figure 2 are the locations of two cut points defined by the instantaneous purities Pu and Pd, where P is the mass fraction of desired component. It should be noted that on-line species-specific detection for determining cut points is particularly useful in displacement chromatography since the formation of bands in close proximity to each other makes nonspecific detection (e.g., the monitoring of UV absorbance) ineffective. Note also that fixed values for Pu and Pd will in general not correspond to a fixed value for the purity of the product fraction, which is presumed to be measured off-line and will be denoted by the symbol Pp. For example, if the concentration of the desired component in the feed tank to the preparative column is reduced or if the relative sensitivity of the monitoring method to the impurities decreases, the values of Pu and Pd used to define cut points would have to be increased if the desired average purity for the entire product fraction is to be maintained. 

Statistical process control methods are applied to preparative chromatography for the case where effluent cut points are determined by online species-specific detection. In particular, Equation 14 (with the quantity in... 

Min, H.L., Nan, W.S., Ming, H.Y., Mei, L.W. and Youk, M.C. (1998) A rapid method for direct determination of free cholesterol in lipids. J. Chinese Agr. Chem. Soc., 36, 123—133. Montiel-Sosa, J.F., Ruis-Pesini, E., Montoya, J., Roncales, P., Lopez-Perez, M.J. and Perez-Martos, A. (2000) Direct and highly species-specific detection of pork meat and fat in meat products by PCR amplification of mitochondrial DNA. J. Agr. Food Chem., 48, 2829—2832. 

Strain or species specific detection, so that distribution patterns can be discerned for individual types, but therefore does not allow an enumeration of total abundance for all similar physiological types. The necessity for cultivation of the antigenic strain also guarantees that most of the strains that are important in the ocean cannot be tested for cross reactivity and it seems likely that they are not detected by these assays. 

SI 1. Stambach, M. N., Falkow, S., and Tompkins, L. S., Species-specific detection of Legionella pneumophila in water by DNA amplification and hybridization. J. Clin. Microbiol. 27, 1257-1261 (1989). 

The existing legislative requirements place further demands on the anal3Tical techniques required for effective enforcement. There is a requirement to increase the degree of test specificity essentially from the level of being able to detect the animal species component of a meat product (see Chapter 7) to the level of the individual tissue component present from that animal. In essence, tissue-specific rather than species-specific detection is what is required to enable accurate discrimination between skeletal muscle tissue and nonmuscle tissue components present in a food product. 

Seyboldt C, John A, Mueffling TV, Nowak B, Wenzel S (2003). Reverse transcription polymerase chain reaction assay for species-specific detection of bovine central nervous system tissue in meat and meat products. J. Food Prot., 66 644—651. 

C. P. Sun, J. C. Liao, Y. H. Zhang, V. Gau, M. Mastali, J. T. Babbitt, et ah Rapid, species-specific detection of uropathogen 16S rDNA and rRNA at ambient temperature by dot-blot hybridization and an electrochemical sensor array. Mol. Genet. Metab. 84, 90-99 [2005]. 

A large number of analytical techniques and strategies have been developed to tackle modern speciation problems. There are four main approaches possible computational, direct species-specific detection, hybrid techniques, and physicochemical characterization techniques. 

Laser desorption/Iaser post-ionisation (two-shot LDI) can display either non-specific detection (using non-resonant single-photon ionisation) or species-specific detection (using resonance-enhanced multiphoton ionisation). By exercising both of these options, complicated mixtures can be analysed for surface species. As efficiency of mass spectral analysis is greatly enhanced by ionising the... 

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