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

The adoption of molecular genetic techniques has greatly improved our ability to characterize microbial populations in situ, leading to more accurate assessments of pollutant biodegradation and the impact of remediation technologies. Initially, biases associated with culturing were avoided by using DNA hybridization techniques with probes containing the catabolic gene of interest. Cross-hybridization at low stringency (46) and exclusion of related but not identical genes at high stringency (15, 30, 56) may produce false-positive and false-negative results with environmental samples. PCR amplification with primers based on conserved regions within the gene of interest, however, will permit amplification despite variability in the overall sequence and detect genotypes not detected by hybridization alone. Molecular methods, particularly in quantitative PCR (Q-PCR), continue to advance rapidly. Our laboratory described a competitive Q-PCR technique to enumerate catechol 2,3-dioxygenase genes as a means to evaluate bioremediation of aromatic hydrocarbons at petroleum-contaminated sites (35). Here we present primer sets targeting additional aromatic catabolic genes and the use of real-time PCR for quantification to extend our ability to enumerate genotypes involved in biodegradation of aromatic pollutants. The two main approaches to Q-PCR are competitive and real-time PCR. In competitive PCR, a comparison of the intensities of the standard and target amplicons allows quantification (42). One limitation of this method is that amplification efficiency of the target and standard are not always equivalent. Furthermore, post-PCR analysis of the products is labor-intensive and not amenable to the large-scale screening of environmental samples. Real-time PCR offers an accurate, sensitive method of quantification without the labor-intensive postamplification analysis and assumptions required by competitive PCR. Detection of product during amplification can be accomplished by several means, including molecular beacons (55), hybridization probes (60), and BODIPY FL-labeled probes and primers (27), but hydrolysis probes and DNA-binding dyes are the most commonly reported methods. The hydrolysis probe (TaqMan) assay takes advantage of the 5′-nuclease ability of DNA polymerase to hydrolyze a labeled probe bound to its target amplicon to produce a signal. The SYBR green assay relies on fluorescence signal produced as the dye binds to double-stranded DNA during the extension step. While the hydrolysis probe provides an additional degree of specificity, identifying an additional highly conserved region for the probe may not always be possible. Furthermore, both the probe and the primers must be from a highly conserved region of the target to avoid biasing quantitation (2). Simpler methods using DNA-staining dyes, such as SYBR green I, have been successful (36, 62), do not require the design of an internal hybridization probe, and thus can be used with any established PCR primers with only minor modifications of the described protocols. This study was conducted to develop multiplex and real-time PCR procedures to quantify aromatic catabolic genes in environmental samples and eventually improve our understanding of biodegradation in the field. The large subunit of aromatic oxygenase genes was chosen as the indicator gene, because it has been implicated in substrate specificity and is one of the rate-limiting steps in aromatic hydrocarbon biodegradation (14) and its DNA sequence is conserved for oxygenases targeting the same substrate. Alignments were constructed from groups of related oxygenase genes, and each primer set was chosen from a conserved region unique to that group of oxygenases. Thus, a single primer set will detect an entire subfamily of related oxygenase genes rather than a species-specific catabolic gene. In all, PCR primer sets which targeted biphenyl dioxygenase, naphthalene dioxygenase, toluene dioxygenase, toluene/xylene monooxygenase, phenol monooxygenase, and ring-hydroxylating toluene monooxygenase genes were identified. Testing and optimization with genetically well-characterized bacterial strains demonstrated the specificity of each primer set. Multiplex PCR protocols were developed to permit simultaneous detection of aromatic oxygenase genes and facilitate rapid screening of environmental samples. Real-time PCR with SYBR green I was used to determine the gene copy number with a quantification limit of 2 × 102 copies of target per reaction mixture. The primer sets and real-time PCR methods presented will be used to assess natural attenuation, investigate the ecology of contaminated sites, and aid in optimization of bioremediation in the field.