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Evaluation of PCR primers for denaturing gradient gel electrophoresis analysis of fungal communities in compost
Author(s) -
Marshall M.N.,
Cocolin L.,
Mills D.A.,
VanderGheynst J.S.
Publication year - 2003
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1046/j.1365-2672.2003.02062.x
Subject(s) - temperature gradient gel electrophoresis , primer (cosmetics) , biology , compost , microbiology and biotechnology , polymerase chain reaction , 16s ribosomal rna , genetics , bacteria , gene , chemistry , ecology , organic chemistry
Aims: Three previously published fungal specific PCR primer sets, referred to as the NS, EF and NL primer sets, were evaluated for use in compost microbial community analysis by PCR and denaturing gradient gel electrophoresis (DGGE). Methods and Results: Primers were first evaluated based on their tolerance to PCR inhibitors. Due to its sensitivity to inhibitors, the NS primer set was determined to require a 10‐fold smaller volume addition of compost DNA to PCR than the EF and NL primer sets, based on a logistic regression model for a 75% PCR success rate. Further evaluation of the EF and NL primer sets involved testing the resolution of PCR products from pure fungal cultures on DGGE. The NL primer set, which targets the more variable 28S rDNA, resulted in multiple bands for each pure culture. Thus, the EF primer set was used to monitor the microbial community during compost colonization studies, where three fungi were inoculated onto autoclaved grape pomace and rice straw compost. Conclusions: Of the three primer sets evaluated, the EF primer set was determined to be the best for PCR–DGGE of compost fungal populations; however, concerns with the EF primer set included the lack of sequence divergence in the targeted region of 18S rDNA and PCR artifacts which interfered with detection of inoculated fungi in the colonization studies. Significance and Impact of the Study: There are many factors related to PCR primers that need to be assessed prior to applying PCR–DGGE to fungal communities in complex environments such as compost.