Open AccessAssessment of the specificity of B urkholderia and P seudomonas qPCR assays for detection of these genera in soil using 454 pyrosequencing
Author(s) -
Bergmark Lasse,
Poulsen Pernille Hasse Busk,
AlSoud Waleed Abu,
Norman Anders,
Hansen Lars Hestbjerg,
Sørensen Søren Johannes
Publication year - 2012
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2012.02601.x
Subject(s) - pyrosequencing , primer (cosmetics) , biology , burkholderia , 16s ribosomal rna , pseudomonas , polymerase chain reaction , computational biology , microbiology and biotechnology , gene , genetics , bacteria , chemistry , organic chemistry
In this study, two highly specific quantitative PCR assays targeting the bacterial genera B urkholderia and P seudomonas were developed and evaluated on soil samples. The primers were targeting different multivariate regions of the 16 S rRNA gene and designed to be compatible with quantitative PCR and the high throughput 454 pyrosequencing technique. The developed assays were validated using the standard methods. All tests with the new developed assays showed very high specificity. Pyrosequencing was used for direct analysis of the PCR product and applied as a specificity measurement of the primers. The P seudomonas primers showed a 99% primer specificity, which covered 200 different P seudomonas sequence clusters in 0.5 g of soil. In contrast to that the same approach using the genus‐specific B urkholderia primers showed only 8% primer specificity. This discrepancy in primer specificity between the normal procedures compared with pyrosequencing illustrates that the common validation procedures for quantitative PCR primers may be misleading. Our results exemplify the fact that current 16 S RNA gene sequence databases might lack resolution within many taxonomic groups and emphasize the necessity for a standardized and functional primer validation protocol. A possible solution to this could be to supplement the normal verification of quantitative PCR assays with a pyrosequencing approach.