Primer and probe sets for group‐specific quantification of the genera Nitrosomonas and Nitrosospira using real‐time PCR

Abstract

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.

Diazotrophic diversity, nifH gene expression and nitrogenase activity in a rice paddy field in Fujian, China

“…Numerous PCR primers targeting the nifH genes have been designed with different range specificity, from "universal" covering different diazotropic taxa (Demba Diallo et al 2008;Poly et al 2001;Zehr et al 1997;Wu et al 2009), to genus and group specific (Huang et al 1999;Olson et al 1998). Recently, quantitative PCR (qPCR) has become a frequently used method to quantify specific genes or microbial groups in different environmental habitats (Lim et al 2008;Novinscak et al 2007). However, few studies have previously been performed on soil where qPCR is combined with reverse transcriptase (RT) PCR, enabling in vitro quantification of specific gene expression (Cook and Britt 2007;Jacobsen and Holben 2007) and qRT-PCR applied to the field emerge as a powerful tool for quantification of an active N 2 -fixing population within a complex community (Church et al 2005) or to monitor diurnal gene expression (El-Shehawy et al 2003;Zehr et al 2007).…”

Community Analysis of Ammonia Oxidizing Bacteria through Molecular Genetics in Activated Sludge of Effluent Treatment Plant

“…Although activated sludge is a common process for wastewater treatment, nitrification failure unfortunately occurs frequently in many WWTPs [4,5], since nitrifiers, especially AOB, grow very slowly, and they are highly sensitive to several environmental and engineering factors, including temperature, pH, Dissolved Oxygen (DO), and a wide variety of chemical inhibitors [6,7]. Therefore, a better understanding of the microbial ecology of AOB in WWTPs could potentially improve the nitrification stability [8]. Culture-dependent methods are biased by the selection of species which obviously do not represent the real dominance structure, and hence give a poor understanding of AOB community structure [9].…”

Agreement between amoA Gene-Specific Quantitative PCR and Fluorescence In Situ Hybridization in the Measurement of Ammonia-Oxidizing Bacteria in Activated Sludge

“…Most notably, quantitative real-time PCR (qPCR) has greatly simplified the quantification of nucleic acids and has been extensively used across a wide range of disciplines and environments (5,10,15,16). Quantitative PCR offers many putative advantages beyond rapid sample processing, such as a linear range exceeding 4 orders of magnitude (13,15,17), high precision (Ͻ2% standard deviation [15]), and high sensitivity (Ͻ5 copies …”

“…Most notably, quantitative real-time PCR (qPCR) has greatly simplified the quantification of nucleic acids and has been extensively used across a wide range of disciplines and environments (5,10,15,16). Quantitative PCR offers many putative advantages beyond rapid sample processing, such as a linear range exceeding 4 orders of magnitude (13,15,17), high precision (Ͻ2% standard deviation [15]), and high sensitivity (Ͻ5 copies [15]). In addition, the specificity of the amplification reaction (e.g., the domain level down to the species level) and the gene to be targeted (e.g., a taxonomic or a functional gene) can be easily controlled by the choice of oligonucleotides (12,17,18).…”

“…Quantitative PCR offers many putative advantages beyond rapid sample processing, such as a linear range exceeding 4 orders of magnitude (13,15,17), high precision (Ͻ2% standard deviation [15]), and high sensitivity (Ͻ5 copies [15]). In addition, the specificity of the amplification reaction (e.g., the domain level down to the species level) and the gene to be targeted (e.g., a taxonomic or a functional gene) can be easily controlled by the choice of oligonucleotides (12,17,18). Using TaqMan probes instead of SYBR green further increases the specificity and sensitivity of qPCR (13,17).…”