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.

41 Reads
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background The global area under brinjal cultivation is expected to be 1.85 million hectare with total fruit production about 32 million metric tons (MTs). Brinjal cultivars are susceptible to a variety of stresses that significantly limit productivity. The most important biotic stress is caused by the Brinjal fruit and shoot Borer (FSB) forcing farmers to deploy high doses of insecticides; a matter of serious health concern. Therefore, to control the adverse effect of insecticides on the environment including the soil, transgenic technology has emerged as the effective alternative. However, the reports, regarding the nature of interaction of transgenic crops with the native microbial community are inconsistent. The effect of a Bt transgenic brinjal expressing the bio-insecticidal protein (Cry1Ac) on the rhizospheric community of actinomycetes has been assessed and compared with its non-transgenic counterpart. Results Significant variation in the organic carbon observed between the crops (non-Bt and Bt brinjal) may be due to changes in root exudates quality and composition mediated by genetic attributes of Bt transgenic brinjal. Real time quantitative PCR indicated significant differences in the actinomycetes- specific 16S rRNA gene copy numbers between the non-Bt (5.62-27.86) × 1011 g-1 dws and Bt brinjal planted soil (5.62-24.04) × 1011 g-1 dws. Phylogenetic analysis indicated 14 and 11, actinomycetes related groups in soil with non-Bt and Bt brinjal crop, respectively. Micrococaceaea and Nocardiodaceae were the dominant groups in pre-vegetation, branching, flowering, maturation and post-harvest stage. However, Promicromonosporaceae, Streptosporangiaceae, Mycobacteriaceae, Geodermatophilaceae, Frankiaceae, Kineosporaceae, Actisymmetaceae and Streptomycetaceae were exclusively detected in a few stages in non-Bt brinjal rhizosphere soil while Nakamurellaceae, Corynebactericeae, Thermomonosporaceae and Pseudonocardiaceae in Bt brinjal counterpart. Conclusion Field trails envisage that cultivation of Bt transgenic brinjal had negative effect on organic carbon which might be attributed to genetic modifications in the plant. Changes in the organic carbon also affect the actinomycetes population size and diversity associated with rhizospheric soils of both the crops. Further long-term study is required by taking account the natural cultivar apart from the Bt brinjal and its near-isogenic non-Bt brinjal with particular reference to the effects induced by the Bt transgenic brinjal across different plant growth stages.
    BMC Microbiology 05/2013; 13(1):122. DOI:10.1186/1471-2180-13-122 · 2.73 Impact Factor
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Activity of ammonia-oxidizing bacteria (AOB) to simultaneous variation in Zn(2+) concentration (0.01-3.5mg/L), temperature (23-33°C), and AOB concentration (3-30 × 10(6)gene copies/mL) in a steel industry wastewater treatment plant was evaluated. Two equations were developed to describe the lag period (i.e., AOB acclimation) and ammonia oxidation rate (i.e., growth of the AOB) depending on the variables. AOB concentration and temperature both had significant effects on lag period and the ammonia oxidation rate. Zn(2+) concentration only had a significant effect on ammonia oxidation rate at 5% α-level. There was a significant interaction between AOB concentration and temperature for both lag period and ammonia oxidation rate. The effects of the variables were not significant when AOB concentration was higher than 2.0 × 10(7)copies/mL. There was no visible shift or changes in AOB communities based on DGGE analysis with amoA gene primers.
    Bioresource Technology 03/2011; 102(5):4196-203. DOI:10.1016/j.biortech.2010.12.035 · 4.49 Impact Factor
  • Source
    • "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) "
    [Show abstract] [Hide abstract]
    ABSTRACT: Actinomycetes degrade cellulose and solubilize lignin during composting. Changes in the diversity of the actinomycetal communities and the 16S rDNA copy numbers of actinomycetes were monitored by denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR), respectively, during continuous thermophilic composting (CTC) and traditional composting (TC). qPCR indicated that the copy numbers from the CTC samples were 25-80% higher than those from the TC samples during similar phases of active composting and they were lower than 3×10(9) gene copies/g (dry weight) in the mature compost from both runs. DGGE showed a more diverse actinomycetal community in the CTC than in TC, averaging 16 bands as compared to 12 bands, at the post peak temperature phase. The study suggested that temperatures higher than 50 °C in CTC benefited the growth of actinomycetes.
    Bioresource Technology 09/2010; 102(2):1383-8. DOI:10.1016/j.biortech.2010.09.034 · 4.49 Impact Factor
Show more


41 Reads
Available from