Primer and probe sets for group-specific quantification of the generaNitrosomonas andNitrosospira using real-time PCR

School of Environmental Science and Engineering, Pohang University of Science and Technology, San 31, Hyoja-dong, Namgu, Pohang, Gyungbuk 790-784, South Korea.
Biotechnology and Bioengineering (Impact Factor: 4.13). 04/2008; 99(6):1374-83. DOI: 10.1002/bit.21715
Source: PubMed


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 beta-proteobacterial AOB in biological nitrification processes and various environments.

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    • "Standard curves were generated by plotting the threshold cycle for each standard, calculated with ABI Prism 7900 SDS 2.2.2 software (Applied Biosystem, USA), against the gene copy number. The amplification efficiency (E) was measured from the slope of the standard curve [29]. The standard curve revealed a slope of – 2.66 corresponding to an efficiency of 137. "
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    • "Germany) with the group-specific primer-probe sets for AOB developed previously (Lim et al., 2008) and adjusted to give a desired initial concentration in each trial. The group-specific probe-primer sets of AOB have been designed to specifically detect subgroups of AOB for cluster of N. europaea, N. nitrosa, N. cryotolerans , and Nitrosospira multiformis). "
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    • "The PCR products were cloned into the Target Clone TM vector (Toyobo, Osaka, Japan). The plasmids were extracted in the range of 10 8 –10 9 copies/lL, serially diluted, and used as templates in qPCR for standard curves generation, as described by Lim et al. (2008) "
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