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Contamination has been an issue plaguing the original PCR experiments. After ordering new solutions and still seeing contamination, we concluded that the problem did not lie with the reagents, the equipment or the graduate students techniques. The problem must have thus been associated with the oligo-primer set developed. There must have been a problem with nonspecific binding of the primers allowing for 75 bp sections of other DNA sequences to be amplified. Also, it was possible that the materials received from the vendors weren’t as specific as we wanted. The primers could have had some additional oligo contained in them, since theoretically one extra sequence present could yield 2n times more products, more than enough to pose PCR contamination problems.
The final target of these studies has always been to develop a highly sensitive assay for the trace detection of prion proteins in body fluids. iPCR, which combines the specificity of immunoassays and the amplification power of PCR, is an ultrasensitive technique for trace analysis of proteins and antigens. iPCR usually lowers the LOD for protein detection about three (3) orders of magnitude, when compared to conventional ELISA. This approach requires: (1) immobilizing a prion protein to a solid support surface; (2) linking thru the detector Ab with a DNA label; (3) amplifying the DNA label by PCR; and (4) subsequently detecting amplification products. The procedure has been been illustrated in Figure 1 (and variations thereof).
One critical aspect of iPCR is the efficient coupling of the target-specific Ab (detector) with the oligo (to form the immunocomplex or chimera). Typically, this was achieved by successive coupling steps of several components. This strategy needs a large number of incubation steps, and usually entails an incomplete interphase coupling of the reagents, occurring with an efficiency of only ~10% for each step. In order to circumvent this problem, we tried to use synthetic strategies based on self-assembly of molecular building blocks to get the immunocomplex (Ab-oligo chimera) for iPCR. This method seemed to be an effective approach to couple several components for iPCR. Its advantage includes high sensitivity, high reproducibility, high linearity, time savings, single step protocol, minimization of signal loss due to inefficient coupling during incubation, and an improved ease of handling. We are not the first to consider using a chimera, but it requires the in-house synthesis, purification, isolation, and characterization before it can be routinely applied in a iPCR format.
Another common problem in immunoassays is nonspecific binding. We performed a series of comparison studies to understand where this came from, and found it mainly occurred between the capture Ab and detector Ab in the immunocomplex formation step. In order to decrease this unwanted binding, we took several steps, such as changing the concentration of capture and detector Ab, trying different blocking buffers and different wash procedures, and so forth. Further studies are in progress.
The stated objectives for this work were: To assess the prevalence, levels, genetic fingerprints and antibiotic susceptibility of L. monocytogenes isolated from the beef-processing environment.
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