Empirical Evaluation of Conditions Influencing the Polymerase Chain Reaction: Enterotoxigenic Escherichia coli as a Test Case
R. du Toit, T. C. Victor, P. D. van Helden
Clinical Chemistry and Laboratory Medicine
False polymerase chain reaction (PCR) results may be obtained under unfavourable reaction conditions. Therefore optimal conditions for the different factors influencing a specific PCR method should be determined before introduction to a clinical diagnostic laboratory. This study has concentrated on the detection of heat-labile enterotoxin-producing E. coli by PCR, with empirical evaluation of various factors. Template was prepared by heat-lysis of E. coli" and this was shown to be adequate for
... CR detection. The results showed that deviation from the optimal conditions of any of the following conditions may lead to false results: lysis of bacterial cells, denaturation temperature during cycling, annealing temperature, primer concentration, enzyme concentration, magnesium concentration and ion concentration. Three different detection methods for PCR product were also evaluated. As little as one bacterium can be detected after 35 cycles of PCR amplification with 32 P^labelled oligonucleotide probe. An alkaline phosphatase-labelled probe was 10-fold less sensitive, whereas 100 bacteria in 10 μΐ of the original sample suspension were necessary to give a positive signal after gel electrophoresis. The information in this study may be useful to those who wish to introduce PCR tests to diagnostic laboratories. unfavourable reaction conditions which influence sen-The polymerase chain reaction (PCR) is an in vitro sitivity, leading to false negative or positive results, method for the amplification of specific nucleic acid In order to avoid false results due to unfavourable sequences by means of repeated cycles of DNA syn-reaction conditions, the factors that may influence thesis (1). This technique has been exploited in the such results have to be critically evaluated beforeâ nalysis of genetic disorders (2), malignant diseases being introduced to a clinical laboratory for routine (2, 3) and in many infectious diseases (4, 5). For many analysis. Some attention has been devoted to the diseases a number of slightly different diagnostic PCR evaluation of factors influencing the PCR reaction (1, methods have been described within a relatively short 10), but it is unlikely that there will be one set of time. Such articles and methods are usually research-optimal amplification conditions for all applications, related and often describe development and testing of Furthermore, it is evident that relatively pure nucleic new primers and analysis of a few samples only (6, acid polymers from eukaryotic cellular genes (11), 7). These methods are not necessarily ready for use viruses (12) and microbes (13), can be used for PCRin routine laboratories, since problems can be en-analysis, but little attention has been given to the countered with contamination (8), the presence of evaluation of conditions influencing the PCR reaction inhibitors of the polymerase enzyme (9), or from when using crude templates.