EMPIRICAL-EVALUATION OF CONDITIONS INFLUENCING THE POLYMERASE CHAIN-REACTION - ENTEROTOXIGENIC ESCHERICHIA-COLI AS A TEST CASE

False polymerase chain reaction (PCR) results may be obtained under un favourable reaction conditions. Therefore optimal conditions for the d ifferent factors influencing a specific PCR method should be determine d 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. Templat e was prepared by heat-lysis of E. coli, and this was shown to be adeq uate for PCR detection. The results showed that deviation from the opt imal conditions of any of the following conditions may lead to false r esults: lysis of bacterial cells, denaturation temperature during cycl ing, annealing temperature, primer concentration, enzyme concentration , magnesium concentration and ion concentration. Three different detec tion methods for PCR product were also evaluated. As little as one bac terium can be detected after 35 cycles of PCR amplification with P-32- labelled oligonucleotide probe. An alkaline phosphatase-labelled probe was 10-fold less sensitive, whereas 100 bacteria in 10 mul of the ori ginal sample suspension were necessary to give a positive signal after gel electrophoresis. The information in this study may be useful to t hose who wish to introduce PCR tests to diagnostic laboratories.