Premium
Primer and probe sets for group‐specific quantification of the genera Nitrosomonas and Nitrosospira using real‐time PCR
Author(s) -
Lim Juntaek,
Do Hyojin,
Shin Seung Gu,
Hwang Seokhwan
Publication year - 2007
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.21715
Subject(s) - taqman , primer (cosmetics) , nitrosomonas , biology , 16s ribosomal rna , in silico , computational biology , oligonucleotide , polymerase chain reaction , ribosomal rna , real time polymerase chain reaction , gene , microbiology and biotechnology , genetics , chemistry , ecology , nitrite , organic chemistry , nitrate
Use of quantitative real‐time PCR (QPCR) with TaqMan probes is increasingly popular in various environmental works to detect and quantify a specific microorganism or a group of target microorganism. Although many aspects of conducting a QPCR assay have become very easy to perform, a proper design of oligonucleotide sequences comprising primers and a probe is still considered as one of the most important aspects of a QPCR application. This work was conducted to design group specific primer and probe sets for the detection of ammonia oxidizing bacteria (AOB) using a real‐time PCR with a TaqMan system. The genera Nitrosomonas and Nitrosospira were grouped into five clusters based on similarity of their 16S rRNA gene sequences. Five group‐specific AOB primer and probe sets were designed. These sets separately detect four subgroups of Nitrosomonas ( Nitrosomonas europaea ‐, Nitrosococcus mobilis ‐, Nitrosomonas nitrosa ‐, and Nitrosomonas cryotolerans ‐clusters) along with the genus Nitrosospira . Target‐group specificity of each primer and probe set was initially investigated by analyzing potential false results in silico, followed by a series of experimental tests for QPCR efficiency and detection limit. In general, each primer and probe set was very specific to the target group and sensitive to detect target DNA as low as two 16S rRNA gene copies per reaction mixture. QPCR efficiency, higher than 93.5%, could be achieved for all primer and probe sets. The primer and probe sets designed in this study can be used to detect and quantify the β‐proteobacterial AOB in biological nitrification processes and various environments. Biotechnol. Bioeng. 2008;99: 1374–1383. © 2007 Wiley Periodicals, Inc.