Competitive PCR-DGGE analysis of bacterial mixtures an internal standard and an appraisal of template enumeration accuracy

Analysis of polymerase chain reaction (PCR) amplified 16S rDNA fragments fr om environmental samples by denaturing gradients of chemicals or heat [dena turing gradient gel electrophoresis (DGGE) and thermal gradient gel electro phoresis (TGGE)] within polyacrylamide gels is a popular tool in microbial ecology. Difficulties in acceptance of the technique and interpretation of the results remain, due to its qualitative nature. In this study we have ad dressed this problem by the construction and evaluation of a quantitative s tandard for incorporation into test DNA samples. The standard was based on a naturally occurring 16S rRNA gene carried by the X-endosymbiont of the ps yllid Anomoneura mori, a gamma-proteobacterium. This sequence is the most A T-rich 16S rDNA gene recovered from any cultured organism or environmental sample described to date, and a specifically amplified rDNA fragment denatu red under exceptionally low stringency denaturing conditions. The native se quence was modified to incorporate perfect matches to the PCR primers used. The efficiency of amplification of this standard in comparison to a range of 16S rDNA sequences and the errors involved in enumerating template molec ules under a range of PCR conditions are demonstrated and quantified. Tests indicated that highly accurate counts of released target molecules from a range of bacterial cells could be achieved in both laboratory mixtures and compost. (C) 2000 Published by Elsevier Science B.V.