Detection and Enumeration of Aromatic Oxygenase Genes by Multiplex and Real-Time PCR

Abstract

Our abilities to detect and enumerate pollutant-biodegrading microorganisms in the environment are rapidly advancing with the development of molecular genetic techniques. Techniques based on multiplex and real-time PCR amplification of aromatic oxygenase genes were developed to detect and quantify aromatic catabolic pathways, respectively. PCR primer sets were identified for the large subunits of aromatic oxygenases from alignments of known gene sequences and tested with genetically well-characterized strains. In all, primer sets which allowed amplification of naphthalene dioxygenase, biphenyl dioxygenase, toluene dioxygenase, xylene monooxygenase, phenol monooxygenase, and ring-hydroxylating toluene monooxygenase genes were identified. For each primer set, the length of the observed amplification product matched the length predicted from published sequences, and specificity was confirmed by hybridization. Primer sets were grouped according to the annealing temperature for multiplex PCR permitting simultaneous detection of various genotypes responsible for aromatic hydrocarbon biodegradation. Real-time PCR using SYBR green I was employed with the individual primer sets to determine the gene copy number. Optimum polymerization temperatures for real-time PCR were determined on the basis of the observed melting temperatures of the desired products. When a polymerization temperature of 4 to 5°C below the melting temperature was used, background fluorescence signals were greatly reduced, allowing detection limits of 2 ؋ 10 2 copies per reaction mixture. Improved in situ microbial characterization will provide more accurate assessment of pollutant biodegradation, enhance studies of the ecology of contaminated sites, and facilitate assessment of the impact of remediation technologies on indigenous microbial populations.

Response of bacterial pdo1, nah, and C12O genes to aged soil PAH pollution in a coke factory area

“…However, it is well known that culture-independent approaches allow for a broader recognition of microbial populations responsible for PAH degradation in environments (Baldwin et al 2003;Jurelevicius et al 2012). Recently, the abundance and composition of PAHdegrading microbial populations present in PAH-polluted environments have been investigated to some extent through direct extraction of DNA from the environment and analysis of PAH-degrading genes (Singleton et al 2005;Lillis et al 2010;Paissé et al 2012).…”

“…Recently, the abundance and composition of PAHdegrading microbial populations present in PAH-polluted environments have been investigated to some extent through direct extraction of DNA from the environment and analysis of PAH-degrading genes (Singleton et al 2005;Lillis et al 2010;Paissé et al 2012). pdo1, nah, and C12O are three important PAH-degrading genes which have been detected widely from environments polluted by aromatic compounds or bacteria isolated from those environments (Baldwin et al 2003;Johnsen et al 2006;Khan et al 2009;Tuan et al 2011). The pdo1 gene encodes the pyrene dioxygenases associated with degradation of high-molecular weight (HMW) PAHs (Johnsen et al 2006), the nah gene encodes naphthalene dioxygenases associated with degradation of low-molecular weight (LMW) PAHs (Baldwin et al 2003), and the C12O gene encodes catechol 1,2-dioxygenase associated with cleavage of the last aromatic ring in the degradative pathway of PAHs (Sei et al 1999).…”

“…pdo1, nah, and C12O are three important PAH-degrading genes which have been detected widely from environments polluted by aromatic compounds or bacteria isolated from those environments (Baldwin et al 2003;Johnsen et al 2006;Khan et al 2009;Tuan et al 2011). The pdo1 gene encodes the pyrene dioxygenases associated with degradation of high-molecular weight (HMW) PAHs (Johnsen et al 2006), the nah gene encodes naphthalene dioxygenases associated with degradation of low-molecular weight (LMW) PAHs (Baldwin et al 2003), and the C12O gene encodes catechol 1,2-dioxygenase associated with cleavage of the last aromatic ring in the degradative pathway of PAHs (Sei et al 1999). Johnsen et al (2006) analyzed nah, phnA, and pdo1 genes using a PCR assay but only detected the presence of nah and pdo1 in soil microcosms spiked with street dust as a PAH source.…”

Development and evaluation of a TaqMan duplex real-time PCR quantification method for reliable enumeration of Candidatus Microthrix

“…The additional TaqMan specificity afforded not only a higher accuracy compared to SYBR Green, but also the possibility of using multiple TaqMan probe and primer sets at the same time (Baldwin et al, 2003(Baldwin et al, , 2008Neretin et al, 2003). Moreover, using mulitplex PCR, simultaneous detection of total Bacteria or other problematic species such as Eikelboom's types (021N, 0675, 0041, 0961, 1701, 0914 and 0092), Thiothrix eikelboomii, Nostocoida limocola, and Gordonia amarae, etc.…”

“…On the other hand, the TaqMan chemistry has an additional specificity as it utilizes a fluorescently labeled probe (Livak et al, 1995). In addition the use of different fluorophores enables the development of multiplex qPCR protocols whereby different targets can be co-amplified and quantified within one single reaction (Baldwin et al, 2003(Baldwin et al, , 2008Neretin et al, 2003). An example of the latter is the use of an exogenous internal positive control (IPC) (Hartman et al, 2005).…”