Polymerase chain reaction species-specific

Samson-Himmelstjerna, G. von, Woidtke, S., Epe, C. and Schnieder, T. (1997) Species-specific polymerase chain reaction for the differentiation of larvae from Diclyocaulus viviparus and Dictyocaulus eckerti. Veterinary Parasitology 68, 119-126. 

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. 

Williams, K. J., Dennis, J. L, Smyl, C., and WaUwork, H. (2002). The application of species-specific assays based on the polymerase chain reaction to analyse Fusarium crown rot of durum wheat. Australas. Plant Pathol. 31,119-127. 

There are various methods for detecting Mycoplasma contamination of cell culture. A sensitive polymerase chain reaction test with broad specificity for Mycoplasma species is our method of choice (8). There are several products available for the eradication of Mycoplasma species from cell lines. The effectiveness of the treatment will depend on the cells and involves trail and error. This is because some cell lines are very sensitive to the chemicals used to eradicate Mycoplasma and may become static or die during treatment. 

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).

Fig. 10. Different techniques for competitive immuno-polymerase chain reaction of small-molecule compounds. (A) Using biotinylated haptens and a DNA-streptavidin nanocircle conjugate [105], hapten-DNA conjugates were synthesized and used for a competitive assay in a sample containing free hapten and capture antibody-coated surfaces [92]. (B) Hapten-coated microplates were simultaneously incubated with a sample containing free hapten and a hapten-specific antibody. Following competitive coupling, the immobilized antibody was subsequently detected by a species-specific antibody-DNA conjugate [93, 94].

L. dispar eggs. Small PTTH-specific nucleotide sequences have been amplified from L. dispar cDNA by the polymerase chain reaction (PCR), and fragments of the expect size have been obtained. The development of bioassays for L. dispar PTTH and recent work on PTTH in other insect species are discussed. 

Nucleic acid analysis. Methods that analyse the microbial community from nucleic acid composition are based on use of the polymerase chain reaction (PCR). Universal forward and reverse primers are used in combination with PCR to amplify species-specific DNA fragments (usually the 16S subunit of ribosomal DNA) from samples isolated directly from soil. Samples then are separated... 

F5. Fries, J. W., Patel, R. J., Piessens, W. F., and Wirth, D. F., Genus- and species-specific DNA probes to identify mycobacteria using the polymerase chain reaction. Mol. Cell. Probes 4, 87-105 (1990). 

More sensitive is the polymerase chain reaction (PCR). Using two PCR primers, even small amounts of DNA can be detected, due to selective amplification (Newton and Graham, 1997). Thus, PCR allows the unambiguous identification of animal and plant species in food or feedstuffs. Moreover, the unique specificity, selectivity, and sensitivity of PCR affords the analysis of complex matrices (Meyer et al., 1993). In addition, primers (in contrast to antibodies) as starting points for PCR are independent of commercial sources and easily accessible to everyone. 

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. 

Related mRNAs encoding various proteins can be detected by different types of in situ hybridization. For this method, the number of specific mRNAs detectable per tumor sample is limited. However, the advantage of in situ hybridization is the same as in immunohistochemistry where the morphology of the tumor is still visible. Specific mRNA-species can be detected by northern blot, nuclease protection assay or reverse transcription (RT) combined with polymerase chain reaction (PCR). Using the modern real-time PCR protocols, reliable quantification of PCR targets is possible. A more complex approach is possible by using the micro-array technology, where hundreds or even more of mRNAs can be detected simultaneously in a semi-quantitative fashion. 

Another important application arising from the work with trichodiene synthase has been the development of Tri5 specific polymerase chain reaction-based assays for the detection of potential trichothecene-producing Fusarium species in pure culture and in contaminated grain (269,275,276). 

Methods for meat species identification based on DNA analysis benefit from the heat stability of the DNA molecule and its high specificity. Originally, DNA methods consisted of immobilization of partially purified and denatured DNA, extracted from the meat product sample, on a nylon membrane, followed by hybridization of a species-specific segment of labeled (colorimetric, fluorescent, or chemiluminescent) DNA with any complementary sequences of DNA present on the membrane. More recently, a DNA amplification method - the polymerase chain reaction - has been used, but this is a relatively expensive and technically demanding technique. 

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